Type I interferon is required for T helper (Th) 2 induction by dendritic cells
2017; Springer Nature; Volume: 36; Issue: 16 Linguagem: Inglês
10.15252/embj.201695345
ISSN1460-2075
AutoresLauren M. Webb, Rachel J. Lundie, Jessica G Borger, Sheila Brown, Lisa M. Connor, Adam N.R. Cartwright, Annette Dougall, Ruud H. P. Wilbers, Peter C. Cook, Lucy H. Jackson‐Jones, Alexander Phythian‐Adams, Cecilia Johansson, Daniel M. Davis, Benjamin G Dewals, Franca Ronchese, Andrew S. MacDonald,
Tópico(s)Immune Response and Inflammation
ResumoArticle17 July 2017Open Access Transparent process Type I interferon is required for T helper (Th) 2 induction by dendritic cells Lauren M Webb Lauren M Webb orcid.org/0000-0002-1903-7570 Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Rachel J Lundie Rachel J Lundie Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK Search for more papers by this author Jessica G Borger Jessica G Borger Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK Search for more papers by this author Sheila L Brown Sheila L Brown Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Lisa M Connor Lisa M Connor Malaghan Institute of Medical Research, Wellington, New Zealand Search for more papers by this author Adam NR Cartwright Adam NR Cartwright Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Annette M Dougall Annette M Dougall Fundamental and Applied Research in Animals and Health, Immunology-Vaccinology, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium Search for more papers by this author Ruud HP Wilbers Ruud HP Wilbers Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands Search for more papers by this author Peter C Cook Peter C Cook Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Lucy H Jackson-Jones Lucy H Jackson-Jones Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK Search for more papers by this author Alexander T Phythian-Adams Alexander T Phythian-Adams orcid.org/0000-0003-0426-6562 Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Cecilia Johansson Cecilia Johansson Respiratory Infection Section, National Heart and Lung Institute, Imperial College London, London, UK Search for more papers by this author Daniel M Davis Daniel M Davis Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Benjamin G Dewals Benjamin G Dewals Fundamental and Applied Research in Animals and Health, Immunology-Vaccinology, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium Search for more papers by this author Franca Ronchese Franca Ronchese orcid.org/0000-0001-5835-8230 Malaghan Institute of Medical Research, Wellington, New Zealand Search for more papers by this author Andrew S MacDonald Corresponding Author Andrew S MacDonald [email protected] Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Lauren M Webb Lauren M Webb orcid.org/0000-0002-1903-7570 Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Rachel J Lundie Rachel J Lundie Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK Search for more papers by this author Jessica G Borger Jessica G Borger Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK Search for more papers by this author Sheila L Brown Sheila L Brown Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Lisa M Connor Lisa M Connor Malaghan Institute of Medical Research, Wellington, New Zealand Search for more papers by this author Adam NR Cartwright Adam NR Cartwright Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Annette M Dougall Annette M Dougall Fundamental and Applied Research in Animals and Health, Immunology-Vaccinology, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium Search for more papers by this author Ruud HP Wilbers Ruud HP Wilbers Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands Search for more papers by this author Peter C Cook Peter C Cook Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Lucy H Jackson-Jones Lucy H Jackson-Jones Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK Search for more papers by this author Alexander T Phythian-Adams Alexander T Phythian-Adams orcid.org/0000-0003-0426-6562 Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Cecilia Johansson Cecilia Johansson Respiratory Infection Section, National Heart and Lung Institute, Imperial College London, London, UK Search for more papers by this author Daniel M Davis Daniel M Davis Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Benjamin G Dewals Benjamin G Dewals Fundamental and Applied Research in Animals and Health, Immunology-Vaccinology, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium Search for more papers by this author Franca Ronchese Franca Ronchese orcid.org/0000-0001-5835-8230 Malaghan Institute of Medical Research, Wellington, New Zealand Search for more papers by this author Andrew S MacDonald Corresponding Author Andrew S MacDonald [email protected] Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK Search for more papers by this author Author Information Lauren M Webb1,7,‡, Rachel J Lundie2,8,‡, Jessica G Borger2, Sheila L Brown1, Lisa M Connor3, Adam NR Cartwright1, Annette M Dougall4, Ruud HP Wilbers5, Peter C Cook1, Lucy H Jackson-Jones2, Alexander T Phythian-Adams1, Cecilia Johansson6, Daniel M Davis1, Benjamin G Dewals4, Franca Ronchese3 and Andrew S MacDonald *,1 1Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester, UK 2Institute of Immunology and Infection Research, Centre for Immunity, Infection and Evolution, University of Edinburgh, Edinburgh, UK 3Malaghan Institute of Medical Research, Wellington, New Zealand 4Fundamental and Applied Research in Animals and Health, Immunology-Vaccinology, Faculty of Veterinary Medicine, University of Liege, Liege, Belgium 5Plant Sciences Department, Laboratory of Nematology, Wageningen University and Research Centre, Wageningen, The Netherlands 6Respiratory Infection Section, National Heart and Lung Institute, Imperial College London, London, UK 7Present address: Baker Institute for Animal Health, Cornell University College of Veterinary Medicine, Ithaca, NY, USA 8Present address: Biomedicine Discovery Institute, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Vic., Australia ‡These authors contributed equally to this work *Corresponding author. Tel: +44 161 275 1504; E-mail: [email protected] The EMBO Journal (2017)36:2404-2418https://doi.org/10.15252/embj.201695345 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Type 2 inflammation is a defining feature of infection with parasitic worms (helminths), as well as being responsible for widespread suffering in allergies. However, the precise mechanisms involved in T helper (Th) 2 polarization by dendritic cells (DCs) are currently unclear. We have identified a previously unrecognized role for type I IFN (IFN-I) in enabling this process. An IFN-I signature was evident in DCs responding to the helminth Schistosoma mansoni or the allergen house dust mite (HDM). Further, IFN-I signaling was required for optimal DC phenotypic activation in response to helminth antigen (Ag), and efficient migration to, and localization with, T cells in the draining lymph node (dLN). Importantly, DCs generated from Ifnar1−/− mice were incapable of initiating Th2 responses in vivo. These data demonstrate for the first time that the influence of IFN-I is not limited to antiviral or bacterial settings but also has a central role to play in DC initiation of Th2 responses. Synopsis Type I interferon (IFN-I) mediates conventional dendritic cell (cDC) activation in a type 2 inflammatory response to parasitic worm infection or the allergen house dust mite, supporting T-helper 2 (Th2) cell induction. cDCs respond to type 2 antigens by producing IFN-I and expressing interferon-stimulated genes. Ifnar1−/− cDCs display reduced phenotypic activation and cytokine production in response to type 2 antigens, indicating that cDC responsiveness to IFN-I is required for this process. Ifnar1−/− cDCs display reduced migration in vivo, indicating that cDC responsiveness to IFN-I is required for this process. Ifnar1−/− cDCs display reduced ability to initiate Th2 responses in vivo, indicating that cDC responsiveness to IFN-I is required for this process. Introduction Although both helminth infection and allergies exert a devastating global impact and lack effective vaccines or refined therapeutics, basic understanding of the key cell types and mediators that initiate and control type 2 immunity is limited. Despite the potential for innate immune cells such as group 2 innate lymphoid cells to influence type 2 responses (McKenzie et al, 2014), it is DCs that are critical for activation and polarization of Th2 immunity (Hammad et al, 2010; Phythian-Adams et al, 2010; McKenzie et al, 2014). However, the signals that DCs provide to facilitate Th2 polarization remain unclear (MacDonald & Maizels, 2008; Bouchery et al, 2014). IFN-I is most well known for its pro-inflammatory role in antiviral immunity (Hoffmann et al, 2015). However, this large family of cytokines (including 14 IFNα subtypes in mice, as well as IFNβ) exert diverse effects in a range of infection settings (Bouchery et al, 2014; McNab et al, 2015). All IFN-I subtypes bind to a common receptor expressed on immune, stromal, and epithelial cells, and studies using IFN-I receptor-deficient mice (Ifnar1−/−) indicate that IFN-I signaling can either enhance or impair the inflammatory response during viral and bacterial infection, depending on context (Ivashkiv & Donlin, 2014; McNab et al, 2015). Many IFN-I effects are mediated by a direct impact on DC phenotype and functionality. For example, IFN-I responsiveness controls the ability of CD8α+ cDC1s to cross-present viral and tumor Ag to CD8+ T cells (Diamond et al, 2011; Pinto et al, 2011; Ivashkiv & Donlin, 2014) and also influences DC activation, migration, and T cell priming in vitro (Parlato et al, 2001; Montoya, 2002; Mattei et al, 2009; Diamond et al, 2011; Pinto et al, 2011). Although it has been suggested that IFN-I may be produced by DCs exposed to live Schistosoma mansoni eggs (Trottein et al, 2004), the role of IFN-I in type 2 inflammation, and how DC function may be modulated by this cytokine family during the orchestration of Th2 responses, is unknown. We have investigated the key factors involved in DC activation and function during Th2 induction using in vitro-generated murine Flt3-L BMDCs (FLDCs), which reflect the heterogeneity and complexity of DC subsets in vivo (Naik et al, 2005), and using in vivo models of Th2 priming. These studies indicate that in addition to producing IFN-I in response to helminth Ag and allergens, DC-intrinsic IFN-I signaling is required for their effective migration, localization, and Th2 induction in vivo. For the first time, we establish a central role for IFN-I as an early positive regulator of Th2 immunity and demonstrate that IFN-I signaling is essential for optimal DC function during type 2 priming. Results An IFN-I signature and Th2 induction by FLDCs To date, studies have failed to identify a defined DC phenotypic activation profile in response to Th2-polarizing helminths (MacDonald & Maizels, 2008; Bouchery et al, 2014). Previous work in this area has predominantly been carried out using murine GM-CSF-derived BMDCs (GMDCs) or cell lines (MacDonald et al, 2001; Trottein et al, 2004). However, FLDCs better represent DC subsets in vivo (Naik et al, 2005; Helft et al, 2015), generating CD24hi equivalents of CD8α+ cDC1s, CD11b+ cDC2s, and plasmacytoid DCs (pDCs; Fig 1A; Brasel et al, 2000; Gilliet et al, 2002; Guilliams et al, 2014). FLDCs were cultured overnight with the potent Th2-inducing soluble egg Ag from S. mansoni (SEA; MacDonald & Maizels, 2008), or heat-killed Salmonella typhimurium (St) as a Th1/17 control (Perona-Wright et al, 2012; Cook et al, 2015). Surprisingly, FLDCs secreted significant levels of IFN-I in response to SEA, including IFNα3 and IFNβ, in excess of that induced by St (Fig 1B). IFN-I production by FLDCs responding to Th2-Ag was not restricted to SEA, but was also evident following their exposure to the allergen house dust mite (HDM; Fig 1C). Despite significant IFN-I induction, SEA failed to stimulate production of the inflammatory mediators IL-1β, IL-6, IL-12p40, IL-12p70, or TNFα, or regulatory IL-10, from FLDCs (Fig 1D), as reported previously by our group and others using GMDCs or human PBMC DCs (MacDonald et al, 2001; de Jong et al, 2002). In stark contrast, St induced high-level production of these cytokines (Fig 1D). Although it has recently been suggested that DC-derived IL-10 and IL-33 are important inducers of Th2 responses (Williams et al, 2013), we were unable to detect significant levels of either IL-10 (Fig 1D) or IL-33 (Fig EV1A) following FLDC exposure to SEA. Figure 1. FLDCs produce IFN-I in response to Th2 Ag A. FLDC subsets were defined by expression of CD11c, CD45R (B220), CD11b, and CD24. pDCs were identified as CD45R+ CD11clo; cDCs are CD11c+ CD45R− with cDC1 and cDC2 subsets. B, C. IFN-I production as measured by ELISA on supernatants from bulk cultures cultured in medium alone (M) or stimulated with 25 μg/ml soluble egg Ag from Schistosoma mansoni (SEA) (B, C), 5 μg/ml Salmonella typhimurium (St) (B), or 50 μg/ml house dust mite (HDM) extract (C). D. Production of DC cytokines from bulk cultures following exposure to SEA or St. E–G. IFN-I production as measured by ELISA on supernatants from bulk cultures cultured in medium alone (M) or stimulated with 1:500 live or dead whole S. mansoni eggs (E), 0.5 μg/ml recombinant omega-1 (F), or FACS-isolated subsets after stimulation with SEA (G). H. Percentage of IFNα6GFP+ DCs after stimulation with SEA, 1 μg/ml CpG, or HDM. I. IFNα3 production by WT, Myd88−/−, and Trif−/− bulk FLDCs after exposure to SEA. Data information: Results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). Data from one of three or more experiments (n = 3 replicate wells per group). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. FLDCs' responses to Ag A. IL-33 production by FLDCs cultured in medium alone (M) or with Ag. B. WT FLDCs were cultured at a ratio of 1 live or dead egg: 500 DCs, or in medium alone (M), for 6 h for gene expression analysis (normalized against Gapdh, a.u.). C, D. DC phenotype following overnight culture in medium alone (M, black), SEA (blue), or St (red). Isotype control (Iso, gray-shaded). E, F. gMFI of surface markers for cDC1 and cDC2 FLDC subsets. G. CD301b expression of FLDC subsets following 18-h culture in the presence or absence of Ag. Data information: Results are mean ± SEM (A, E, F) (one-way ANOVA) or least squares mean ± SEM (B) (analyzed using a three-way full-factorial fit model, with contrast analysis used to test differences between experimental groups). ***P < 0.001, ****P < 0.0001. Data from one of three or more experiments (n = 3 replicate wells per group) (A, C–G) or three experiments pooled (n = 7–8 replicate wells) (B). gMFI, geometric mean fluorescence intensity. a.u., arbitrary units. Download figure Download PowerPoint In order to assess whether IFN-I induction by the soluble extract SEA was also evident with whole S. mansoni eggs, we cultured FLDCs with live or dead eggs (Fig 1E). This demonstrated significant induction of both IFNα3 and IFNβ following FLDC exposure to dead eggs, as well as a clear IFN-I signature in the form of upregulated IFN-stimulated genes (ISGs), including Ifit1, Mx1, and Oas1a (Fig EV1B). Co-culture of FLDCs with live eggs showed only a trend toward IFN-I secretion and ISG induction, suggesting that the components responsible for DC IFN-I production are predominantly released from dead, or dying, eggs. In addition, IFN-I production was also detected in FLDCs exposed to the T2 ribonuclease (RNase) omega-1 (Everts et al, 2009; Wilbers et al, 2017), which is the major Th2 immunostimulatory factor in, and one of the most abundant components of, S. mansoni egg secretions (Cass et al, 2007; Fig 1F). To identify which FLDC subset(s) produced IFN-I in response to Th2-inducing Ag, cells were sorted prior to SEA stimulation, demonstrating that the primary source was cDC1s (Fig 1G). This was unexpected, given that pDCs are a key source of this cytokine, particularly in early viral infection (Swiecki & Colonna, 2010). As a complementary approach, we cultured FLDCs generated from mice that express GFP when IFNα6 is expressed (Kumagai et al, 2007) with SEA, CpG, or HDM (Fig 1H). These IFNα6 reporter cells confirmed that in addition to cDC1-restricted production of IFNα3 (Fig 1G), both cDC1s and cDC2s had the capacity to produce IFNα6 in response to SEA. They also demonstrated that pDCs did not respond to the Th2-associated Ags SEA or HDM with upregulation of IFN-I production and that, although HDM promoted FLDC IFNα3 and IFNβ (Fig 1C), no IFNα6 was induced in either cDCs or pDCs by this allergen. As expected, CpG was the most effective IFNα6 stimulus in all three FLDC subsets. Together, this shows that cDCs, and particularly cDC1s, can produce IFN-I in response to both helminth Ag and allergens, as has been reported for antiviral settings (Diebold et al, 2003; Kato et al, 2005). cDC1s in vivo produce IFN-I in response to TLR3-TRIF agonists such as polyI:C (Miyake et al, 2009), while pDCs primarily produce IFN-I following TLR7 or TLR9 stimulation in a MyD88-dependent manner (McNab et al, 2015). In agreement with this, we found that FLDC production of IFN-I in response to SEA was TRIF-dependent, not MyD88-dependent (Fig 1I). In fact, IFN-I induction was negatively regulated by MyD88 (Fig 1I), a phenomenon reported for TRIF-dependent cytokine responses in macrophages (Johnson et al, 2008), but not previously identified as a mechanism of regulating TRIF-dependent IFN-I production in DCs. In addition to their impact on DC cytokine secretion, SEA and St upregulated expression of surface molecules associated with Ag presentation and costimulation (MHC II, CD40, and CD86) in both cDC1s and cDC2s (Fig EV1C and E), while pDC phenotypic activation was not dramatically altered by either Ag (Fig EV1C). It has recently been suggested that expression of PD-L2 and CD301b may be typical of DCs activated with Th2-inducing Ag (Gao et al, 2013; Kumamoto et al, 2013). Although upregulation of PD-L2 was observed on cDCs responding to SEA, increased expression of this marker was not restricted to Th2 Ag, but was also evident on DCs responding to St (Fig EV1D and F). Neither Ag influenced the low level of CD301b expressed by any FLDC subset (Fig EV1G). Notably, in most cases, the degree to which activation molecules were upregulated was less striking with SEA than St (Fig EV1). This muted cDC surface activation following exposure to SEA is in keeping with previous reports of GMDCs or human PBMC DCs exposed to SEA in vitro, or splenic and hepatic DCs from S. mansoni-infected mice, and may reflect an alternative, and more restricted, DC activation phenotype that is common to Th2 settings (MacDonald et al, 2001; de Jong et al, 2002; Straw et al, 2003; MacDonald & Maizels, 2008; Lundie et al, 2016). In order to act as effective Ag-presenting cells (APCs), DCs must be able to migrate to the dLN (Alvarez et al, 2008). Since the role of the different DC subsets in Th2 priming is currently unclear (MacDonald & Maizels, 2008; Bouchery et al, 2014), and FLDC ability to generate Th2 responses has not yet been addressed, we next assessed their capacity to migrate effectively, and to initiate and polarize immune responses after adoptive transfer into naïve mice. Following injection of dsRed+ FLDCs, cDCs capably trafficked to the dLN (Fig 2A and B). Transferred CD45R+ pDCs could not be detected in the dLN (Fig 2A), in agreement with previous work demonstrating that pDCs can only gain entry to a LN via the blood through inflamed high endothelial venules and do not routinely migrate via the lymphatics (Diacovo, 2005). While the proportions of cDC1s and cDC2s were approximately 50:50 in cultures prior to transfer (as in Fig 1A), the majority of DCs reaching the dLN were cDC2s (Fig 2A and C), consistent with the suggestion that cDC2s are the primary DC subset that polarizes Th2 responses in vivo (Tussiwand et al, 2015). Interestingly, even though SEA-exposed cDCs maintained a muted activation phenotype post-transfer (Fig 2D), a characteristic that has also been identified in vivo during S. mansoni infection (Straw et al, 2003), they competently migrated to the dLN T cell zones (Fig 2E). In addition, despite their limited activation profile, and significant IFN-I production, SEA-exposed FLDCs effectively promoted Th2 polarization following transfer, with clear induction of IL-4, IL-5, IL-10, and IL-13 in dLN restimulations (Fig 2F). As in many Th2 settings (Pearce & MacDonald, 2002), transferred FLDCs also induced SEA-specific IFNγ. It is possible that the minor population of cDC1s present in the dLN were responsible for the low-level IFNγ response that was seen in this setting, as has been reported with the in vivo equivalents of this subtype (Everts et al, 2016). In keeping with their inflammatory phenotype, St-exposed FLDCs capably induced recipient IL-10, IL-17, and IFNγ production (Fig 2G). Consistent with previous reports, no IL-17 was detectable following SEA-activated DC transfer (Larkin et al, 2012; Cook et al, 2015) and no Th2 cytokines were evident following St-activated DC transfer. Figure 2. FLDCs migrate to the dLN and induce Ag-specific responses following adoptive transferFollowing Ag stimulation, dsRed+ FLDCs were injected s.c. into naïve WT mice, and 48 h later, dLNs were harvested and the presence of dsRed+ CD11c+ cells was assessed by flow cytometry (A, B) or confocal microscopy (E). A. dsRed+ CD11c+ FLDCs were gated as pDCs (CD45R+), cDC1s (CD45R− CD24+), or cDC2s (CD45R− CD11b+). B. Absolute numbers of dsRed+ CD11c+ FLDCs in the dLN calculated from flow analysis and cell counts. C. Percentage of transferred dsRed+ CD11c+ cells that were cDC1s or cDC2s. D. CD86 expression on transferred dsRed+ CD11c+ FLDCs. E. Confocal microscopy of dLN sections after FLDC transfer: Upper row depicts overlay of CD3 (green), CD45R (gray) and dsRed (red); bottom row depicts dsRed (red) alone. White dashed line represents division between T cell (CD3+) and B cell zones (CD45R+). Scale bars represent 38 μm. F, G. Seven days after transfer, dLN cells were restimulated for 72 h with 15 μg/ml SEA (F), 1 μg/ml St (G), or medium alone (M) and cytokine production assessed by ELISA. Data information: Results are least squares mean ± SEM (B–D) or mean ± SEM (F, G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (analyzed using a three-way full-factorial fit model, with contrast analysis used to test differences between experimental groups (B–D) or one-way ANOVA (F, G)). Data from one of three or more experiments (A, E–G) or three experiments pooled (B–D) (n = 3–9 animals per group). gMFI, geometric mean fluorescence intensity. Download figure Download PowerPoint FLDCs depend on IFN-I signaling for Th2 induction Having established that SEA triggered FLDC IFN-I secretion (Fig 1), while also conferring Th2-initiation ability (Fig 2), we next addressed the importance of IFN-I production in the Th2 induction process. Given that DCs themselves can be a target of IFN-I (Montoya, 2002; Mattei et al, 2009), we first determined whether IFN-I-responsiveness was required for optimal DC function following SEA stimulation. To this end, we used Ifnar1−/− mice to generate FLDCs lacking the IFNAR1 subunit of the IFN-I receptor, thus rendering them unresponsive to IFN-I (Hwang et al, 1995). Ifnar1−/− FLDCs displayed significantly reduced expression of the ISGs Ifit1 and Mx1 in response to SEA (Fig 3A), and markedly impaired secretion of IFN-I (Fig 3B), consistent with previous reports using Ifnar1−/− fibroblasts in non-Th2 settings (Marié et al, 1998). Additional aspects of SEA-induced activation of WT cDC1s and cDC2s were dramatically impaired in Ifnar1−/− FLDCs, with reduced expression of all markers measured (Fig 3C). IFN-I signaling has been implicated in controlling many aspects of DC function, not limited to surface activation and cytokine production, but also regulating their survival and turnover (Mattei et al, 2009). However, we did not identify any defect in either differentiation, or survival, of Ifnar1−/− FL-cDCs (Fig EV2). Most strikingly, in the absence of IFNAR1, the ability of FLDCs to promote SEA-specific Th2 responses in vivo was completely ablated, while IFNγ induction by these DCs was not significantly affected (Fig 3D). Further, using IL-4 reporter mice (Mohrs et al, 2005) as recipients, we found that Ifnar1−/− SEA-activated FLDCs displayed impaired ability to prime CD4+ T cell IL-4 secretion (huCD2) or mRNA expression (eGFP) in vivo (Fig 3E). Together, our data demonstrate for the first time that IFN-I responsiveness is essential for optimal DC activation by Th2-inducing Ag and is a key factor governing DC Th2 priming ability in vivo. Figure 3. FLDCs depend on IFN-I signaling for optimal activation and Th2 induction A–D. WT or Ifnar1−/− FLDCs were cultured with 25 μg/ml SEA or in medium alone (M) for 6 h for gene expression analysis (A) (normalized against Gapdh, a.u.) or 18 h for analysis of IFN-I production (B), surface phenotype (C), or injection s.c. into naïve WT mice (D). (D) Seven days after transfer, dLN cells were restimulated for 72 h with 15 μg/ml SEA or medium alone and cytokine production assessed by ELISA (medium-stimulation-alone values subtracted). E. WT or Ifnar1−/− FLDCs were transferred s.c. into KN2xIL-4eGFP mice; 7 days later, the presence of IL-4+ (huCD2+, IL-4 protein) and IL-4eGFP+ (IL-4 mRNA) CD4+ T cells was assessed by flow cytometry. Data information: Results are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way ANOVA). Data from one of three or more experiments (n = 3 replicate wells (A–C) or 5 animals (D, E) per group). a.u., arbitrary units. gMFI, geometric mean fluorescence intensity. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Ifnar1−/− FLDCs display normal development and viability FLDCs were cultured for 18 h in medium alone and analyzed for subsets by flow cytometry. Following 18-h culture, FLDCs were stained with 7AAD and Annexin V to assess viability of cDC populations. 7AAD+ Annexin V+ were classed as late apoptotic, 7AAD− Annexin V+ as early apoptotic, and 7AAD− Annexin V− as live cells. Results are mean ± SEM. Data information: Data from one of three or more experiments (n = 3 replicate wells per group). Download figure Download PowerPoint cDCs depend on IFN-I signaling for effective migration to, and localization within, the dLN To determine why Ifnar1−/− FLDCs were impaired in their Th2 induction ability, we first assessed their capacity to process and present Ag to OVA-specific OT-II CD4+ T cells. T cell proliferation was comparable in co-cultures containing WT or Ifnar1−/− cDCs with either OVA protein (OVA) or peptide (pOVA) indicating that Ifnar1−/− cDCs had no defect in their capacity to capture, process, or present Ag in vitro (Fig 4A and B). Supporting unimpaired Ag uptake and processing in the absence of IFNAR responsiveness, Ifnar1−/− cDCs internalized and processed DQ-OVA as effectively as WT (Fig 4C). Further demonstrating that Ifnar1−/− cDCs displayed no fundamental deficiency in their ability to polarize CD4+ T cells in vitro, both WT and Ifnar1−/− cDCs co-cultured with IL-4, IL-13, or IL-10 reporter CD4+ T cells (Mohrs et al, 2005; Kamanaka et al, 2006; Neill et al, 2010; Cook et al, 2015) effectively induced T cell expression of huCD2 (IL-4; Fig 4D), IL-10eGFP (Fig 4E), and IL-13eGFP (Fig 4F) under polarizing conditions. These in vitro experiments indicate that DC Ag uptake, processing, and presentation were not significantly impaired in the absence of IFNAR1 signaling. Figure 4. Ifnar1−/− FL-cDC APC fun
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