Regulatory T (Treg) cells establish tolerance, prevent inflammation at mucosal surfaces, and regulate immunopathology during infectious responses. Recent studies have shown that Delta-like ligand 4 (Dll4) was upregulated on APC after respiratory syncytial virus (RSV) infection, and its inhibition leads to exaggerated immunopathology. In the present study, we outline the role of Dll4 in Treg cell differentiation, stability, and function in RSV infection. We found that Dll4 was expressed on CD11b+ pulmonary dendritic cells in the lung and draining lymph nodes in wild-type BALB/c mice after RSV infection. Dll4 neutralization exacerbated RSV-induced disease pathology, mucus production, group 2 innate lymphoid cell infiltration, IL-5 and IL-13 production, as well as IL-17A+ CD4 T cells. Dll4 inhibition decreased the abundance of CD62LhiCD44loFoxp3+ central Treg cells in draining lymph nodes. The RSV-induced disease was accompanied by an increase in Th17-like effector phenotype in Foxp3+ Treg cells and a decrease in granzyme B expression after Dll4 blockade. Finally, Dll4-exposed induced Treg cells maintained the CD62LhiCD44lo central Treg cell phenotype, had increased Foxp3 expression, became more suppressive, and were resistant to Th17 skewing in vitro. These results suggest that Dll4 activation during differentiation sustained Treg cell phenotype and function to control RSV infection.
This article is featured in In This Issue, p.1379
Notch is a highly conserved signaling pathway that contributes to cell differentiation and function. Engagement of Notch receptors (Notch 1–4) and Notch ligands (Delta-like ligand 1, 3, 4, Jagged 1, 2) initiates cleavage of the Notch intracellular domain. The Notch intracellular domain binds to recombination binding protein (RBP)-Jκ and activates the transcription of Notch target genes in association with a coactivator of the mastermind-like (MAML) family (MAML1–3). Notch signaling regulates various stages of T cell lineage development and differentiation, including early stages of thymocyte development, as well as CD4 Th differentiation (1, 2). In the priming stage of Th cell differentiation, Notch ligand Delta-like ligand 4 (Dll4) sensitized Ag responses and augmented T cell activation (3), whereas separate studies demonstrated that Dll4 and Notch could activate production of IFN-γ and the Th1 transcription factor T-bet (2–5). During Th2 differentiation, Jagged1 and intracellular Notch promoted Gata3 and Il4 expression (2, 6, 7), whereas Dll4 suppressed Il4, Il5, and Il13 (8, 9). Furthermore, Dll4 and Notch were reported to promote Th17 and Th9 differentiation by enhancing Il17a, Rorc, and Il9 expression (10, 11). Taken together, Notch can serve as an amplifier for persistent Th1, Th2, and Th17 cell differentiation (4), suggesting that it is not a skewing signal, but rather that it enhances coactivation. However, the role of Dll4 and Notch signaling in regulatory T (Treg) cells remains unresolved. Jagged2 and specific receptors, Notch1 and Notch3, promoted the Treg cell master transcription factor Foxp3 expression and Treg cell survival (12–16), and RBP-Jκ was reported to directly bind the Foxp3 promoter and regulate Foxp3 transcription (16). In contrast, inactivating Notch signaling after Foxp3 is expressed enhanced Treg cell numbers and promoted tolerance (17). Blockade of Notch receptors and Notch ligands expanded Foxp3+ T cell populations in vivo in experimental autoimmune encephalomyelitis, graft-versus-host disease (GVHD), and type 1 diabetes (18–21). However, the role of Notch ligands in Treg cell development and their resistance to inflammation during infection has not been well defined.
Notch ligands can be induced on APC by pathogen-associated molecular patterns (2, 6). Pathogens themselves can also induce Notch ligands. Studies showed that respiratory syncytial virus (RSV) induced Dll4 expression on dendritic cells (DC) in vitro (8, 22), and Dll4 blockade exacerbated RSV-induced Th2 airway pathogenesis (8). Because Treg cells are required to limit pulmonary inflammation and pathogenic Th2 responses during RSV infections (23–25), we hypothesized that initial exposure of Dll4 may modulate peripherally induced Treg (iTreg) cell differentiation, homeostasis, and stability to control the intensity of the immune response and lung pathology during RSV infection. In the present study, we report that Dll4 sustained CD62LhiCD44lo central Treg (cTR) cells and solidified iTreg cell identity during infection. This study defines novel roles of Dll4 in iTreg cell subset regulation and iTreg cell stability.
Materials and Methods
Six- to eight-week-old female BALB/cJ and C57BL/6J mice were purchased from The Jackson Laboratory. Female CD45.1 (B6-Ly5.1/Cr) mice were purchased from Charles River Laboratories. Foxp3eGFP mice (B6.Cg-Foxp3tm2(EGFP)Tch/J, stock no. 006772) were bought from The Jackson Laboratory and bred in-house. CD4-specific double-negative MAML (DNMAML) mice (Cd4-Cre × R26DNMAMLf) and CD4-specific Rbpj knockout mice (Cd4-Cre × Rbpjf/f) were generated as described (26–28). All mice were housed in the University Laboratory Animal Facility under animal protocols approved by the Animal Use Committee at the University of Michigan.
RSV infection and in vivo neutralization of Dll4
RSV line 19 was a clinical isolate originally from a sick infant in the University of Michigan Health System to mimic human infection (29). BALB/cJ mice were anesthetized and infected intratracheally (i.t.) with 1 × 105 PFU of line 19 RSV, as previously described (8). For Dll4 blockade in vivo, 2.5 mg of purified polyclonal anti-Dll4 Ab or control IgG were injected i.p. 2 h before RSV infection at day 0. The same doses of control or anti-Dll4 Ab were given on days 2, 4, and 6.
Left lobe of lung was fixed with 4% formaldehyde and embedded in paraffin, and 5 μm of section were stained with periodic acid–Schiff (PAS) to detect mucus.
RNA isolation and quantitative PCR
RNA were extracted with TRIzol (Invitrogen) by following the manufacturer’s protocol, and 1 μg of total RNA was reverse transcribed to cDNA to determine gene expression using TaqMan gene expression primer/probe sets. Dll1, Dll4, Jag1, and Jag2 were detected by SYBR as described (2). Delta4 primers were 5′-AGGTGCCACTTCGGTTACACAG-3′ and 5′-CAATCACACACTCGTTCCTCTCTTC-3′. Muc5ac and Gob5 expression was assessed by custom primers as described (30). Detection was performed in an ABI 7500 real-time PCR system (Applied Biosystems). Gene expression was calculated using the ΔΔCt method and normalized with Gapdh as input control.
Primary cells isolation and cytokine production assay
Mice lungs were chopped. Lung and mediastinal lymph nodes (mLN) were enzymatically digested using 1 mg/ml collagenase A (Roche) and 25 U/ml DNaseI (Sigma-Aldrich) in RPMI 1640 with 10% FCS for 45 min at 37°C. Tissues were further dispersed through an 18-gauge needle (10-ml syringe) and filtered through 100-μm nylon mesh twice. Cells (5 × 105) from mLN cells were plated in 96-well plates and restimulated with 105 PFU of RSV line 19 for 48 h. IFN-γ, IL-4, IL-5, IL-13, IL-17A, IL-10, and IL-9 levels in supernatants were measured with a Bio-Plex cytokine assay (Bio-Rad Laboratories).
Extracellular and intracellular flow cytometry analysis
Single-cell suspensions of lung and lymph nodes were stimulated with 100 ng/ml PMA, 750 ng/ml ionomycin, 0.5 μl/ml GolgiStop (BD Biosciences), and 0.5 μl/ml GolgiPlug (BD Biosciences) for 5 h (when mentioned). After excluding dead cells with Live/Dead fixable yellow stain (Invitrogen), cells were preincubated with anti-FcγR III/II (BioLegend) for 15 min and labeled with the following Abs from BioLegend: anti-B220 (RA3-6B2), CD3 (145-2C11), CD4 (GK1.5), CD8α (53-6.7), CD11b (M1/70), CD11c (N418), CD25 (PC61), CD44 (IM7), CD45 (30-F11), CD62L (MEL-14), CD69 (H1.2F3), CD103 (2E7), CD127 (SB/199), CCR7 (4B12), Dll1 (HMD1-3), Dll4 (HMD4-1), Gr-1 (RB6-8C5), I-A/I-E (M5/114.15.2), and ST2 (DIH9). For innate lymphoid cell staining, lineage markers were anti-CD3, CD11b, B220, Gr-1, and TER119. After 30 min of incubation at 4°C, cells were washed and proceed to intracellular staining.
For intracellular staining, cells were fixed and permeabilized with a transcription factors staining buffer set (eBioscience). Cells were labeled with Abs from eBioscience: Foxp3 (FJK-16s), IL-17A (eBio17B7), IL-13 (eBio13A), GATA3 (TWAJ), retinoic acid–related orphan receptor (ROR)γt (AFKJS-9), and GzmB (NGZB) for 30 min at room temperature. Flow cytometry data were acquired with an LSR II (BD Biosciences) or NovoCyte (ACEA Biosciences) flow cytometer and were analyzed with FlowJo software (Tree Star).
Naive CD4 T cell isolation and stimulation
CD4+CD25−CD62LhiCD44lo naive T cells were enriched from spleen using a naive CD4 T cell isolation kit (Miltenyi Biotec) with >92% purity. Naive T cells were then plated and cultured in 24-well plates. Naive T cells (106/0.5 ml) were stimulated with plate-bound anti-CD3 (2.5 μg/ml; eBioscience), soluble anti-CD28 (3 μg/ml; eBioscience), and plate-bound recombinant Dll4 (1.65 μg/ml or the dose mentioned; R&D Systems); to skew toward in vitro iTreg cells, human TGF-β1 (2 ng/ml; R&D System) and mouse IL-2 (10 ng/ml; R&D Systems) were added at the same time; to restimulate toward in vitro–induced Th17 cells, mouse IL-6 (10 ng/ml; R&D Systems), human TGF-β1 (2 ng/ml; R&D Systems), anti–IFN-γ neutralizing Ab (10 μg/ml; eBioscience), anti–IL-4 neutralizing Ab (10 μg/ml; eBioscience), and anti–IL-12/23 p40 neutralizing Ab (10 μg/ml; eBioscience) were added at 5 × 105/0.2 ml of viable iTreg culture or 105/0.2 ml sorted enhanced GFP (eGFP)+ iTreg cells when mentioned.
Cell sorting and in vitro suppression assay
Single-cell sorting was performed on a FACSAria II (BD Biosciences). DAPI−CD4+eGFP+ viable Treg cells were sorted with >93% efficiency. A suppression assay was performed as described with small modifications (31). In brief, naive T cells isolated from CD45.1 mice were labeled with CellTrace Violet (CTV) (Invitrogen). Labeled CD45.1+ naive T cells (2.5 × 104) were cocultured with serial-diluted eGFP+ Treg cells in 96-well round-bottom plates. Dynabeads mouse T activator CD3/CD28 (0.625 μl; Invitrogen) was added to 0.2 ml of culture. After 72 h, cells were harvested and CD45.1+ responder cell proliferation was accessed by CTV dilution.
Data were analyzed by Prism 6 (GraphPad Software). Data presented are mean values ± SEM. Comparison of two groups was performed with an unpaired, two-tailed Student t test. Comparison of three or more groups was analyzed by ANOVA with a Tukey posttest. A p value <0.05 was considered significant.
Upregulation of Dll4 during RSV infection on CD11b+ pulmonary DC
Previously published data have shown that Dll4 expression was upregulated on DC after RSV infection in vitro (8, 22). To further characterize the expression of Dll4 during RSV infection in vivo, BALB/cJ mice were i.t. infected with 1 × 105 PFU of RSV before harvesting of lung tissues at 2 and 6 d postinfection (dpi). Dll4 transcripts were upregulated significantly at 6 dpi, whereas transcripts for the other Notch ligands Dll1, Jag1, and Jag2 were not increased in the lung (Fig. 1A). After gating on MHChiCD11c+ DC (Fig. 1B), Dll4 expression level was increased consistently on MHC class II (MHC-II)hiCD11c+ DC after 6 dpi (Fig. 1C). To further understand which type of DC expressed Dll4 after RSV infection, two populations of pulmonary DC were identified to determine Dll4 expression: MHC-IIhiCD11c+CD11b+CD103− DC and MHC-IIhiCD11c+CD103+CD11b− DC. CD11b+ DC expressed Dll4, whereas few of the CD103+ DC expressed Dll4 during RSV infection (Fig. 1D). When we examined the number of DC in lung and draining lymph node at 6 dpi, increasing numbers of Dll4+CD11b+ DC in both lung and mLN were detected as the infection progressed. In contrast, the number of Dll4+CD103+ DC was significantly lower and variable in lung or mLN postinfection (Fig. 1E). These data indicated that Dll4 was expressed on pulmonary DC, especially CD11b+ DC, after RSV infection in vivo.
Inhibition of Dll4 exacerbated RSV-induced immunopathology in vivo
Next, we explored the role of Dll4 in controlling RSV immunopathology by blocking Dll4 in vivo during RSV infection. We have previously demonstrated that our polyclonal anti-Dll4 Ab is specific for Dll4 and not other Notch ligands (8). The polyclonal anti-Dll4 Ab decreased the abundance of Notch target gene Hes1 transcripts in activated T cells (Th0) in coculture with fixed Dll4-expressing stromal cells (Supplemental Fig. 1A), indicating that anti-Dll4 Ab blocked Dll4-mediated Notch activation in vitro. We next aimed at blocking Dll4 in vivo by injecting 2.5 mg of purified anti-Dll4 Ab i.p. Mice were then infected i.t. with RSV 2 h after Ab injection at day 0, followed by i.p. injection of anti-Dll4 every other day. After 8 d, the abundance of Hey1–Notch target gene transcripts was decreased in the lung (Supplemental Fig. 1B) and sorted CD4 T cells (Supplemental Fig. 1C) of anti-Dll4–treated mice, suggesting that systemic administration of anti-Dll4 Abs inhibited Notch signaling activation in CD4 T cells in vivo. Dll4 blockade increased peribronchial infiltration by mononuclear cell clusters and drove mucus production in the airway as assessed in PAS-stained lung sections (Fig. 2A). As an indication of RSV-induced pathology, expression of the mucus-associated gene gob5 (mClca3) was substantially increased (Fig. 2B), but the viral protein mRNA expression measured with RSV-F and RSV-G were not significantly different at either 6 or 8 dpi (Fig. 2C, data not shown). Although Dll4 inhibition increased total CD4 T as described (Fig. 2D) (8), data also showed that more CD4 T cells acquired a CD44hiCD62Llo effector phenotype (Fig. 2E). Another activation marker CD25 was not changed in CD4 T cells at 8 dpi (data not shown). Thus, Dll4 inhibition significantly altered lung immunopathology during RSV infection. Besides CD4 T cells, RSV infection can also expand group 2 innate lymphoid cells (ILC2) between 4 and 6 dpi in lung to be mucogenic and immunopathogenic (32). In this study, we investigate whether Notch ligand Dll4 affects ILC2 homeostasis. Strikingly, Dll4 inhibition increased the number of lineage−CD45+CD127+CD90.2+ST2+CD25+ ILC2 in lung (Fig. 2F) and mLN (Fig. 2G) at 6 dpi. Thus, Dll4 blockade increased ILC2 in the lungs during RSV infection.
Excessive cytokine production is one of the key features leading to RSV pathogenesis. The IFN-γ promotes viral clearance whereas IL-4, IL-5, IL-13, and IL-17A are pathogenic (29–34). In this study, we determined the effect of Dll4 blockade on cytokine profiles in draining lymph nodes. Equal numbers of mLN cells were cultured and restimulated with 105 PFU of RSV. In vivo Dll4 blockade during RSV infection led to significantly increased Th2 cytokines IL-5 and IL-13 as well as IL-17A whereas IFN-γ, IL-4, IL-9, and IL-10 were not significantly affected in RSV-rechallenged lymph node cells (Fig. 2H). To specify whether Dll4 regulated Th cell cytokine production in CD4 T cells, mLN cells were restimulated with PMA plus ionomycin and CD4 T cells were examined by flow cytometry. Dll4 blockade significantly increased IL-13+ and IL-17A+ CD4 Th cells in mLN at both 6 and 8 dpi (Fig. 2I, data not shown). These data indicated that Dll4 neutralization exacerbated RSV-induced immunopathology, as shown by elevated mucus production, activated CD4 T cells, and Th2-Th17 cytokine overproduction.
Dll4 neutralization reduces cTR cells and increases Th17-like Treg cells during RSV infection
Several studies demonstrated that Treg cells quickly accumulate in lymphoid tissues and in the airway to dampen activated T cell infiltration and Th2 responses (23, 24, 35), and RSV infection drove Treg cells to Th2-like effector cells with impaired function (36). Our group showed that Dll4 inhibition exacerbated type 2 cytokine production and activated T cell infiltration, but little is known about the role of Dll4 in Treg cell development, stability, and function. After targeting Dll4 as previously described, total Treg cells did not change in mLN at 6 dpi (Fig. 3A). However, when gating on CD62LhiCD44lo central CD4 T cells (Fig. 3B), Dll4 inhibition decreased Foxp3+ cell frequency and expression (Fig. 3C, 3D) while decreasing expression of the CCR7 chemokine receptor on central T cells (Fig. 3E, 3F). CCR7-mediated signals recruit CD62LhiCD44lo T cells to the T cell zone, and CCR7 is a marker of cTR cells (37). Dll4 neutralization decreased the cTR cell populations (Fig. 3G), whereas effector Treg (eTR) cells were not altered in mLN (Fig. 3H). These data suggested that Dll4 specifically sustained cTR cells in draining lymph node during RSV infection.
In contrast to its effects in lymph nodes, Dll4 neutralization enhanced the abundance of Foxp3+ Treg cells in the lungs of infected mice (Fig. 4A) and CD44hiCD62LloFoxp3+ eTR cells in lung at 8 dpi (Fig. 4B) without affecting cTR cells (data not shown). Based on our previous finding that Dll4 blockade enhanced IL-13 and IL-17A production in CD4 T cells (Fig. 2I), we examined whether Dll4 can also perturb cytokine production in Foxp3+ CD4 T cells. Lung cells harvested at 8 dpi were restimulated and stained for intracellular Foxp3, IL-13, and IL-17A before flow cytometric analysis. In vivo Dll4 blockade significantly increased the abundance of IL-17A+Foxp3+ (Fig. 4C) and IL-13+Foxp3+ (Fig. 4D) but not IL-13−, IL-17A−, Foxp3+ Treg cells (data not shown). To further characterize whether Dll4 regulates either Th17 or Th2 transcription factor coexpression in Treg cells was examined. RORγt or GATA3 was costained with Foxp3 after restimulation. Dll4 neutralization increased the percentage of RORγt+Foxp3lo cells and RORγt+Foxp3− but not RORγt−Foxp3+ after 8 dpi (Fig. 4E, 4F). We did not detect significant changes in either GATA3+ Treg cells or total GATA3+ CD4 T cells in both lung and lymph nodes after Dll4 inhibition (data not shown). These data suggested that Dll4 inhibition drove a Th17-like effector phenotype in Treg cells during RSV infection in vivo.
To further investigate whether Dll4 regulated Treg cell functional markers, several were examined, including ICOS, programmed cell death protein 1, granzyme B (GzmB), and neuropilin-1 (Nrp1). Dll4 inhibition decreased the expression of GzmB, whereas ICOS, programmed cell death protein 1, and Nrp1 expression was unchanged in Foxp3+ Treg cells at 8 dpi (Fig. 4G, data not shown). It was previously shown that during RSV infection, GzmB was the critical functional molecule for Treg cell function to limit the associated immunopathology (25). We found that Dll4 neutralization decreased the frequency of GzmB+ in Foxp3+ Treg cells in the lung after 8 dpi (Fig. 4H), especially for GzmB+ eTR cells (Fig. 4I). Our results indicate that Dll4 prevented Foxp3 Treg cell acquisition of Th17-like effector phenotype, and it supported GzmB expression in Treg cells in the lung during RSV infection.
Dll4 activates and maintains inducible Treg cells in vitro
Our data reveal differential effects of Dll4 in Foxp3+ Treg cells in lymphoid versus nonlymphoid tissue during pulmonary infection. To further investigate Dll4 effects on Treg cell differentiation in a controlled context, splenic CD4+CD25−CD62LhiCD44lo naive CD4 T cells were activated with anti-CD3/anti-CD28 with or without plate-bound recombinant Dll4, and skewed toward Treg cells with TGF-β and IL-2. We first confirmed that Dll4 was able to activate expression of the Notch target gene Hes1 in naive CD4 T cells and Th0 and Treg cells (Supplemental Fig. 2A–C) in a dose-dependent manner (Supplemental Fig. 2C) and activated intracellular domain of Notch1 cleavage (Supplemental Fig. 2D). Subsequently, naive T cells were skewed toward Th0 or iTreg cells. Foxp3 expression was significantly increased with Dll4 activation under iTreg cell skewing conditions (Fig. 5A). Importantly, Dll4 increased CD25+Foxp3+ iTreg cells in a dose-dependent manner only in the presence of TGF-β (Fig. 5B). Furthermore, Foxp3 expression was significantly decreased with homozygous ROSA26-driven expression of the pan–Notch inhibitor DNMAML1 (Fig. 5C), and with inactivation of the Rbpj gene, encoding RBP-Jκ (Fig. 5D). As MAML1 and RBP-Jκ mediate transcriptional activation from Notch signaling, these data revealed that Dll4-activated Foxp3 expression was dependent on canonical RBP-Jκ/MAML–dependent Notch signaling. To further characterize Foxp3+ cells in this differentiation system, primary Dll4-activated iTreg cell cultures were rested in IL-2 for 3 d without Dll4 (Fig. 5E). Dll4-treated iTreg cells consistently maintained a higher percentage of Foxp3+ (Fig. 5F) and higher Foxp3 expression (Fig. 5G). To investigate whether Dll4 affected homeostatic features on Treg cells during in vitro skewing, CD62L and CD44 expression was evaluated before or after the IL-2 rest phase. Naive CD4 T cells that were exposed to Dll4 during primary iTreg cell skewing maintained more CD62LhiCD44loFoxp3+ cells consistently independent of the dose of IL-2 during the rest period (Fig. 5H) and at multiple time points after primary skewing (Fig. 5I). These data suggest that the presence of Dll4 during iTreg cell activation and differentiation stabilized Foxp3 expression and sustained more Treg cells with the CD62LhiCD44lo phenotype in vitro.
Dll4-exposed iTreg cells are more suppressive in vitro
Our data showed that Dll4 inhibition decreased Treg cell functional markers during RSV infection (Fig. 4), and Dll4/Notch directly sustained Foxp3 expression and cTR cells (Fig. 5). These findings imply that Dll4 may change Treg cell function. To further evaluate the function of iTreg cells after Dll4 exposure, we sorted viable, Foxp3-eGFP+ iTreg cells from iTreg cultures on day 6 and cocultured these cells with CD45.1+ naive CD4 T cells as responders. CD45.1+ naive T cells were labeled with CTV and stimulated with anti-CD3/anti-CD28 mouse T activator for 72 h. Viable CD45.1+ cells were gated to examine CTV dilution as readout of cell proliferation (Fig. 6A). Responder cells that were cocultured with Dll4-treated iTreg cells were less proliferative, especially at the 1:1 and 1:2 cell ratio of Treg/Tnaive cells (Fig. 6B), suggesting that Dll4-treated iTreg cells were more suppressive than iTreg cells without Dll4. Intriguingly, CD45.1+ responder cells were less viable when cocultured with Dll4-exposed iTreg cells (Fig. 6C). Because one of the functions of Treg cells is to consume survival signals from other T cells, we evaluated IL-2Rα (CD25) expression after 6 d of iTreg cell skewing. Costimulation of iTreg cells with Dll4 enhanced CD25 expression on Foxp3+ Treg cells in vitro (Fig. 6D). These data indicated that Dll4-exposed iTreg cells were more functional in vitro.
Dll4 and Notch activation strengthened iTreg cells to be less plastic toward Th17
The data above showed that Dll4 inhibition increased Th17-like Treg cells during RSV infection. To examine whether Dll4 and Notch signaling could inhibit acquisition of a Th17 effector phenotype in iTreg cells, naive CD4 T cells were purified and cultured in iTreg cell skewing conditions. Il17a expression was examined during iTreg cell skewing. Inactivation of Notch increased Il17a expression during iTreg cell skewing (Fig. 7A), whereas Foxp3 transcripts were decreased (Fig. 7B). Both DNMAML1 expression and Rbpj inactivation in CD4 T cells led to increased IL-17A in iTreg+ Dll4-skewed cultures (Fig. 7C). These data suggest that intrinsic Notch activation inhibited Il17a expression while enhancing Foxp3 expression in iTreg cells during differentiation. To further investigate whether Dll4 affects IL-17A production after Treg cell differentiation, an equal number of cells were challenged under IL-6 plus TGF-β Th17 conditions for 3 more days after the standard 6 d iTreg cell skewing culture. In Th17 restimulation, Dll4-treated iTreg cells secreted significantly less IL-17A (Fig. 7D) with associated decreased RORγt+ cells (Fig. 7E). Furthermore, Dll4-exposed iTreg cells retained a higher percentage and expression level of Foxp3 (Fig. 7F, 7G). These data suggest that Dll4-mediated signals during iTreg cell differentiation led to less inflammatory Th17 skewing while maintaining Treg cell commitment. To clarify whether Dll4-educated Foxp3+ iTreg cells had less plasticity to become RORγt+, 105 viable Foxp3-eGFP+ iTreg cells were sorted after 6 d of skewing and cultured in Th17 conditions. Approximately 40% of eGFP+ iTreg cells without Dll4 acquired RORγt+ expression, whereas Dll4-skewed eGFP+ iTreg cells expressed significantly less RORγt+Foxp3+ (Fig. 7H). Although a similar amount of eGFP+ cells lost their Foxp3 expression to be Foxp3− in either non-Dll4 or Dll4-educated iTreg cells (data not shown), Dll4-treated iTreg cells still retained more Foxp3+RORγt− cells during Th17 skewing (Fig. 7I). These data suggest that Dll4-educated iTreg cells had less plasticity in an inflammatory Th17 environment.
The objective of this study was to determine whether the Notch ligand Dll4 influenced Foxp3+ Treg cell development, homeostasis, and function during pulmonary infection. Notch signaling has been suggested to be instrumental in Foxp3+ Treg cell formation and maintenance (17, 38). Notch ligands—Dll1, Jagged1, and Jagged2—have been shown to regulate Treg cell expansion and elicit suppressive activity (13, 16, 39). The present study suggests that Dll4 mechanistically regulates the stability of iTreg cells. Previous studies showed that primary RSV infection enhanced Foxp3+ Treg cells in the airway (24, 35). Our data indicate that Dll4 sustained Foxp3 expression especially in cTR cells and altered homeostatic features and functional markers of Treg cells. In vivo Dll4 inhibition allowed the acquisition of effector cytokine production in Treg cells, increased their effector phenotype (CD44hiCD62Llo), and decreased functional Treg cell markers (GzmB) during infection. In vitro skewing of iTreg cells costimulated with Dll4 from naive T cells had enhanced regulatory function, retained their cTR cell phenotype, and were more stable with less susceptibility to become RORγt+ Th17 cells. Importantly, stable and functional Treg cells prevent autoimmunity and enhance immunity against chronic infection (40). Thus, identifying Dll4 as one stabilizing factor in autologous Treg cells could potentially further optimize clinical efficacy of treatment protocols (41, 42).
Dll4/Notch signaling appears to have differential functions in various disease models. Using systemic blockade, Dll4 inhibition ameliorated GVHD, type 1 diabetes, and experimental autoimmune encephalomyelitis (18, 19, 21, 43, 44). In contrast, Dll4 blockade leads to exacerbated allergic airway disease and pulmonary RSV infection responses, as well as to increased bacillus Calmette–Guérin-induced granuloma formation (8, 45). These different results could be reconciled with a revised model that Notch signaling fine-tunes T cell responses to different environmental cues (4), and thereby plays a distinct role in different diseases, such as Ag-driven/infectious versus autoimmune or allogeneic responses. Recently, a novel study demonstrated that intrinsic Notch signaling limited peripheral Treg cell maintenance during steady-state and in GVHD (17). In contrast to these latter findings, our results suggest that Dll4 and canonical Notch signaling sustained iTreg cell differentiation and stability to prevent immunopathogenesis of pulmonary infection. Recent evidence supports that iTreg cells are important for mucosal homeostasis, suppression of immune responses to environmental and food Ags, and to impede mucosal inflammation. In contrast, naturally occurring Treg (nTreg) cells prevent autoimmunity and alter the threshold of immune activation (46, 47). Notch signaling and Dll4 restrain nTreg development in vivo (19), and GVHD was mainly prevented by nTreg (48, 49) but not iTreg cells due to lineage instability (50). To answer the question whether Dll4 differentially affects nTreg versus iTreg cells, we used Nrp1 to distinguish nTreg versus iTreg cells in vivo as described (51–54). Our preliminary findings showed that Dll4 inhibition decreased Nrp1− Treg cells during RSV infection but not Nrp1+ Treg cells in the spleen (data not shown). Therefore, we speculate that Dll4/Notch may have a differential role on nTreg versus iTreg cells, thereby limiting nTreg cells in GVHD but enhance iTreg cells in RSV infection. Additional research needs to be done to elucidate this intriguing hypothesis.
RSV infection can induce IL-17A production (34, 55), and IL-17A facilitates mucus production to promote Th2-driven immunopathology (34). Furthermore, Treg cells could be reprogrammed to Th17-like in the presence of appropriate inflammatory cytokines (56, 57). iTreg cell plasticity is critical for conversion to IL-17A–producing Foxp3+ and RORγt+Foxp3+ populations under multiple microenvironments and diseases (58–60). In the present study, Dll4 prevented IL-17A+ Treg cell development in infected mice. Moreover, Dll4-“educated” Foxp3-GFP+ iTreg cells retained higher Foxp3 expression and resisted skewing toward Th17 in vitro. These results indicate that Dll4 modulates Treg cell stability and plasticity toward Th17 in inflammatory environments.
Pathogenic RSV infection also exhibits a type 2–biased immune response (61). Our group has shown that Dll4 prevented Th2 cytokine secretion in CD4 T cells during RSV infection. Because both Notch signaling and Treg cells have been reported to be involved in Th2 programs, it raised the question of whether Dll4 regulates Th2 cytokine production directly or indirectly through Treg cell effects. Surprisingly, our data did not demonstrate a significant perturbation in GATA3+ Th2 lineage after Dll4 inhibition (data not shown). Instead, our data presented a dysfunctional Treg cell and Th17-like effector phenotype after Dll4 inhibition. Because IL-17A can enhance Th2 responses in RSV infection and other pulmonary inflammation, these results may suggest that the Th2 response is a result of an indirect effect of reduced Treg cell function and increased IL-17.
Besides Th2 cells, recent studies have demonstrated that RSV infection enhances ILC2 with a concurrent IL-13 increase (32). The present study indicated that Dll4 inhibition increased the number of ILC2 corresponding with elevated IL-5 and IL-13 levels. Notch activation has been shown to support ILC2 progenitor development in vitro (38, 62), but the in vivo role of Dll4 in ILC2 biology during infection has not been described. Our novel findings further highlight the importance of Dll4 in ILC2 homeostasis in vivo, but it is not clear whether the regulation is direct or indirect. Because iTreg cells inhibit ILC2 and attenuate pulmonary inflammation (63), future studies will be necessary to specify the indirect contribution of iTreg cells in restraining ILC2 during pulmonary infection.
The homeostatic features of Foxp3+ Treg cells were characterized with CD62L, CD44, and CCR7. CD62LhiCD44loCCR7+ cTR cells are quiescent, long-lived, and dependent on IL-2, whereas CD44hiCD62LloFoxp3+ eTR cells appear to be shorter lived (37, 64). These two subpopulations favored distinct compartments, as cTR cells homing to secondary lymphoid organs were found to be suppressive for autoimmunity (65). Lymph node sensitization is critical to mount a proper immune response because insufficient lymph node formation led to impaired Foxp3 Treg cell function, promoted IL-17A–mediated pulmonary pathology, and initiated lymphoid cell clusters in the lung (66, 67). In the present study, data showed that Dll4 sustained the cTR cell population in secondary lymphoid organs while preventing Th17 effector phenotype during RSV infection, representing a novel role for Dll4 in cTR cell maintenance.
In addition to the alteration of the Treg cell cytokine phenotype, the present study further suggests that Dll4 sustained Treg cell function by influencing GzmB in vivo and CD25 in vitro. Intriguingly, GzmB in Foxp3+ Treg cells controlled RSV-induced T cell infiltration in airways (25), and GzmB has been postulated to be a better functional marker of iTreg cells during viral infection (68). Further investigations are needed to characterize the function of Treg cell subsets during RSV infection. In the present study, our findings with regard to GzmB may provide a mechanistic explanation for Dll4 inhibition in exacerbating RSV-induced CD4 and CD8 T cell infiltration (8).
Taken together, our study demonstrates that Dll4/Notch promotes iTreg cell development, homeostasis, stability/plasticity, and function in vitro and in vivo during RSV infection. Dll4 prevented RSV pathogenesis and supported iTreg cell development in vitro. The effects of Dll4 on Treg cells are an example of how Notch signaling can promote T cell homeostasis and disease regulation. By extending our understanding of Dll4 in Treg cell stability and function, our results may provide a road for future translational research leading to a better therapeutic strategy.
We thank Dr. M.A. Schaller, Dr. D.E. de Almeida Nagata, Dr. Catherine Ptaschinski, and Dr. Ivan P. Maillard for helpful discussions.
This work was supported by National Institutes of Health Grants AI036302 (to N.W.L.) and AI091627 (to I.P.M.).
The online version of this article contains supplemental material.
Abbreviations used in this article:
Delta-like ligand 4
group 2 innate lymphoid cell
MHC class II
mediastinal lymph node
naturally occurring Treg
recombination binding protein
retinoic acid–related orphan receptor
respiratory syncytial virus
The authors have no financial conflicts of interest.