IL-7Rα Expression Regulates Murine Dendritic Cell Sensitivity to Thymic Stromal Lymphopoietin

Laura Kummola, Zsuzsanna Ortutay, Xi Chen, Stephane Caucheteux, Sanna Hämäläinen, Saara Aittomäki, Ryoji Yagi, Jinfang Zhu, Marko Pesu, William E. Paul and Ilkka S. Junttila
J Immunol May 15, 2017, 198 (10) 3909-3918; DOI: https://doi.org/10.4049/jimmunol.1600753

Abstract

Thymic stromal lymphopoietin (TSLP) and IL-7 are related cytokines that mediate growth and differentiation events in the immune system. They signal through IL-7Rα–containing receptors. Target cells of TSLP in Th2 responses include CD4 T cells and dendritic cells (DCs). Although it has been reported that expression of TSLP receptor (TSLPR) on CD4 T cells is required for OVA-induced lung inflammation, DCs have also been shown to be target cells of TSLP. In this study, we show that murine ex vivo splenic DCs are unresponsive to TSLP, as they fail to phosphorylate STAT5, but in vitro overnight culture, especially in presence of IL-4, renders DCs responsive to both TSLP and IL-7. This induced responsiveness is accompanied by dramatic upregulation of IL-7Rα on DCs with little change in expression of TSLPR or of γc. In splenic DCs, the induction of IL-7Rα occurs mainly in CD8− DCs. In vivo, we found that IL-4 has a differential regulatory role on expression of IL-7Rα depending on the cell type; IL-4 decreases IL-7Rα expression on CD4 T cells whereas it upregulates the expression on DCs. Our results indicate that the induction of IL-7Rα expression on DCs is critical for TSLP responsiveness and that IL-4 can upregulate IL-7Rα on DCs.

Introduction

Signaling by thymic stromal lymphopoietin (TSLP) and IL-7 depends on ligand-mediated assemblage of a receptor complex consisting of a cytokine-specific binding chain and an auxiliary chain. The TSLP and IL-7 receptors share IL-7Rα, although they use this chain distinctly. IL-7 binds with high affinity to IL-7Rα, and this complex then binds common γ chain (γc), activating the Jak1/Jak3 kinases, followed by phosphorylation of critical tyrosine residues on IL-7Rα (1). Docking of STAT5 to the phosphorylated tyrosine residues of IL-7Rα results in Jak-mediated phosphorylation and subsequent nuclear translocation, as well as DNA binding of STAT5, resulting in regulation of STAT5-mediated transcription. In contrast, TSLP binds TSLP receptor (TSLPR), a close homolog of γc, followed by IL-7Rα recruitment to the complex. The KD for TSLP binding to TSLPR is ∼7 × 109 (2), whereas the KD for IL-7 binding to IL-7Rα is 1 × 1010 (3), indicating that initial cytokine binding to ligand-binding receptor chain occurs with higher affinity by IL-7 than by TSLP. As opposed to IL-7, the Jak1 and Jak2 kinases are activated by TSLP in human and mouse primary CD4 T cells with the consequent phosphorylation of STAT5 (1). In human myeloid dendritic cells (DCs), activation of STAT6 by TSLP has also been reported (4).

Functionally, IL-7 is a critical growth factor for B and T cell development and induces survival of memory T cells (5). Recently, the role of IL-7 in development of innate lymphoid cells has also been established (6). It is of interest that epithelial cells possess the capacity to produce IL-7, which might be important for survival of tissue lymphocytes locally (7). Memory cell competition for IL-7 has been suggested to be the mechanism of regulation of IL-7–mediated growth (8).

Although mast cells (9), basophils (10), and DCs (11) have been reported to produce TSLP, the main TSLP-producing cells appear to be the epithelial cells in humans and particularly the keratinocytes in mice (12–14). Thus, TSLP appears to be principally expressed at skin and mucosal barriers; in contrast, IL-7 production seems more a property of stromal cells in primary and secondary lymphoid organs (15). Effectively, this suggests a specialization of the functions of the two cytokines and particularly emphasizes the potential importance of TSLP in action at mucosal surfaces, be it in the induction of Ag-presenting competence in DCs or in the differentiation/expansion of memory/effector T cells in tissues.

To respond to TSLP, target cells need to express both IL-7Rα and TSLPR, whereas those responsive to IL-7 require expression of IL-7Rα and γc (16). Of conventional or plasmacytoid DCs from unmanipulated mice, only migratory and skin DCs express IL-7Rα at a low level, although IL-7Rα expression appears to be important for DC development (17). Human myeloid DCs have been reported to alter their behavior in response to TSLP by directly upregulating CD40 and CD80 and by enhancing their capacity to induce T cell proliferation and chemotaxis, with the latter through expression of CCL17 and CCL22 (12, 18). Freshly isolated human myeloid DCs express low levels of both IL-7Rα and TSLPR, and overnight (o/n) culture of these cells strongly upregulates expression of both receptor chains (19). Recently, basophils were shown to be responsive to TSLP, and it has been reported that such basophils acquire a Th2 phenotype in that they produce more IL-4 in response to cytokine stimulation than do basophils cultured in IL-3 (20). IL-4 itself is a critical regulator of Th2 responses, and the cytokine binding receptor chain for IL-4 (IL-4Rα) is found virtually on all cells and, thus, basically all cells have the capability to respond to IL-4, though with different sensitivity (21).

Because IL-7Rα appears to be expressed at very low levels in murine DCs, we became interested in how they could be a direct target of TSLP. We reasoned that upregulation of IL-7Rα on DCs might be required. Indeed, we observed that neither TSLP nor IL-7 can cause freshly isolated splenic DCs to phosphorylate significant amounts of STAT5, but that o/n culture of splenic DCs, especially in the presence of IL-4, results in their responsiveness to both cytokines. We observed that TSLPR and γc were expressed on freshly isolated DCs and showed only modest enhancement as a result of o/n culture, but that IL-7Rα expression was dramatically upregulated by o/n culture, and more prominently so in the presence of IL-4. In spleen, IL-7Rα upregulation was less prominent in CD8+ DCs, whereas CD4+ DCs showed significant upregulation. This suggests that “tuning” of different DC subclasses to TSLP and IL-7 occurs via differential regulation of receptor chains for these cytokines and prepares these cells for differential responsiveness. We also discovered that IL-4 further upregulated IL-7Rα expression and cytokine responsiveness of DCs,suggesting yet another regulatory mechanism for IL-4 in orchestrating allergic responses. Furthermore, our results suggest that TSLP responses require both the induction of TSLP production and the induction of IL-7Rα on DCs, presumably with both events occurring in the same microanatomic location.

Materials and Methods

Mice and cell cultures

C57BL/6 mice (The Jackson Laboratory) were housed either in the National Institute of Allergy and Infectious Diseases pathogen-free animal facility or at the animal facility of the School of Medicine, University of Tampere. Experiments were performed under a protocol approved by the National Institute of Allergy and Infectious Diseases Animal Care and Use Committee. TSLPR−/− mice were provided by Dr. W.J. Leonard (National Heart, Lung, and Blood Institute, National Institutes of Health). The spleens and lymph nodes from 6- to 10-wk-old mice were minced and incubated for 30 min at 37°C in HBSS containing 75 μg/ml Liberase DL and 10 U/ml DNase (both Roche, Basel, Switzerland). The cells were filtered through a 40-μm cell strainer, and RBCs were lysed with 30 s ACK (Lonza, Basel, Switzerland) treatment. Where indicated, DCs were isolated using a MACS pan-DC isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were either cultured o/n in RPMI 1640 with 10% FBS, l-glutamine, and penicillin/streptomycin (Lonza) or used immediately. Where indicated, spleens were mechanically disrupted using a cell culture strainer to release DCs. In the in vivo experiment, mice were injected i.p. twice with either 6 μg of IL-4 (PeproTech, Rocky Hill, NJ) in PBS, complexed with 30 μg of anti–IL-4 Ab (clone BVD4-1D11; Thermo Fisher Scientific, Carlsbad, CA) as described (22), or 30 μg of anti–IL-4 alone in PBS. The injections were given 8 h apart. The mice were euthanized 12 h after the last injection. Human DCs were purified from peripheral blood of five healthy donors under ethical permission R21002 from the Ethics Committee of Pirkanmaa Hospital District using pan-human DC purification kit (Miltenyi Biotec). All reagents used were provided endotoxin free by the manufacturers and where applicable they were dissolved into sterile PBS containing BSA (low endotoxin; Sigma-Aldrich, St. Louis, MO). Strict aseptic techniques in biosafety level 2 sterile tissue culture hoods were used in all experiments.

Abs, cytokines, flow cytometry, and statistics

Mouse Abs and isotype controls were either from BD Biosciences (Franklin Lakes, NJ; pSTAT5, B220), eBioscience (Santa Clara, CA; CD11c, CD49/Dx5, MHC class II [MHC II], CD3, CD4, CD8, F4/80, IL-7Rα, γc, CD80, CD86, isotype controls for IL-7Rα and γc), or R&D Systems (Minneapolis, MN; TSLPR and polyclonal goat Ig as a control). Human Abs were from eBioscience (CD3, CD4, CD8, CD11c, CD14, CD19, CD56, HLA-DR), BD Biosciences (CD80), or Thermo Fisher Scientific (IL-7Rα and isotype control for IL-7Rα). Murine cytokines were from PeproTech (GM-CSF, IL-4, IL-7) or R&D Systems (TSLP). Human cytokines (IL-4, TSLP) were from PeproTech. Where indicated, 1 μg/ml LPS (InvivoGen, San Diego, CA), 20 ng/ml IL-4 with or without LPS, or 40 ng/ml IL-7 was added to the o/n culture. For pSTAT5 assays, cultured cells were starved for 2 h in RPMI 1640 (with l-glutamine, penicillin/streptomycin, and 1% FBS) at 37°C and stimulated for 15 min with 100 ng/ml GM-CSF, IL-7, or TSLP. Intracellular pSTAT5 staining was done after surface staining by permeabilizing the cells with 90% ice-cold methanol (23). For surface staining, 0.1% BSA, 0.1% mouse serum, and CD64 and CD32 blocking 4G2 Abs were used. Cells were analyzed with FACSCanto II (BD Biosciences), and data were analyzed with the FlowJo (Tree Star, Ashland, OR) analysis program. The Prism program (GraphPad Software, La Jolla, CA) was used for statistics. Geometrical means ± SEM are indicated. The p values were calculated using a two-tailed, paired Student t test.

Results

Splenic DCs respond to TSLP after in vitro culture

The TSLP and IL-7 receptors share IL-7Rα (Fig. 1A). We became interested in how TSLP might regulate DC function, particularly because of a recent report showing that splenic or lymph node DCs of mice had no detectable IL-7Rα, with the exception of lymph node migratory DCs, and their dermal and epidermal counterparts showed low levels of IL-7Rα expression (17). Because several reports suggest a role for TSLP in the regulation of DC activities (24), the low level of expression of IL-7Rα in murine DCs seemed enigmatic. We chose to determine whether DCs were responsive to TSLP and IL-7 by measuring the appearance of an immediate signaling intermediate elicited by these cytokines, STAT5 tyrosine phosphorylated at Y594 (pSTAT5). We prepared single-cell suspensions from spleens of C57BL/6 mice by using DNase/Liberase treatment, followed by enrichment of DCs as described in Materials and Methods. The cells were incubated with the indicated cytokines for 15 min or left untreated, surface stained to allow their phenotyping, and permeabilized to allow the detection of pSTAT5. We analyzed the degree of pSTAT5 in CD11cbright, MHC IIbright cells (Fig. 1B). As a positive control for pSTAT5 in DCs, we stimulated the cells with GM-CSF.

FIGURE 1.
FIGURE 1.

Splenic DC IL-7 and TSLP responsiveness requires o/n culture of the cells. (A) Schematic presentation of assemblage of functional IL-7 and TSLP receptor complexes. (B) Gating of enriched CD11cbright/MHC IIbright splenic DCs. (C) STAT5 phosphorylation of DCs stimulated as indicated for 15 min either immediately after harvesting the spleens (ex vivo) or after 16 h of in vitro culture. Representative flow cytometer plots are shown. (D) Quantitation of pSTAT5 in DCs in response to cytokines. The number of pSTAT5+ cells after GM-CSF stimulation was given the value of 100%. The bars represent the average percentage (±SEM indicated) of pSTAT5+ cells among CD11cbright/MHC IIbright cells after cytokine stimulation in six independent experiments. *p < 0.05, **p < 0.01, two-tailed, unpaired t tests. (E) The expression of CD80 and CD86 activation markers was measured from CD11cbright/MHC IIbright cells either immediately after harvesting spleens or after 16 h of culture. Representative result is shown. (F) Quantitation of CD80 and CD86 expression. Statistical analysis of three independent experiments is shown. The bars represent the average of mean fluorescence intensity (MFI; ±SEM indicated). **p < 0.01, ***p < 0.001, two-tailed, paired t tests. FSC, forward scatter; SSC, side scatter.

As expected, stimulation of splenic DCs with GM-CSF resulted in robust phosphorylation of STAT5 (Fig. 1C) in most of these cells. TSLP and IL-7 stimulated a modest or nonexisting response in the freshly isolated splenic DCs (Fig. 1C, 1D). We then asked whether “spontaneous” (i.e., DC damage–independent, microbial product–independent) DC activation caused by o/n culture altered TSLP or IL-7 responsiveness by these cells. We cultured splenocytes o/n on regular tissue culture plates (25) and stimulated them with GM-CSF, TSLP, or IL-7 for 15 min. GM-CSF induced STAT5 phosphorylation on most of the cultured DCs as it had on freshly isolated DCs. In contrast to freshly isolated splenic DCs, a substantial proportion of the o/n-cultured DCs showed robust phosphorylation of STAT5 in response to TSLP or to IL-7 (Fig. 1C, lower panel). Quantitatively, when percentage of GM-CSF–induced pSTAT5 was set to 100, stimulation of splenic DCs with IL-7 or TSLP after o/n culture showed a significant enhancement in the proportion of pSTAT5+ DCs (Fig. 1D). The increase in the proportion of DCs that exhibited STAT5 phosphorylation in response to TSLP or IL-7 was highly significant (p < 0.05 and p ≤ 0.01, respectively, by two-tailed unpaired t test Fig. 1D).

To establish that the DNase/Liberase treatment had not impaired the cytokine response, we compared IL-7 stimulation on ex vivo splenocytes prepared either mechanically or with DNase/Liberase treatment; IL-7 induced pSTAT5 to a similar degree on splenic CD4 T cells from both preparations (Supplemental Fig. 1, upper panel), indicating that IL-7Rα was not degraded by DNase/Liberase. As the DC yields were higher when using DNase/Liberase treatment, we chose to use this approach. Also, enrichment of DCs did not affect expression of IL-7Rα on freshly isolated DCs (Supplemental Fig. 1, lower panel). Overnight culture activated DCs; both CD80 and CD86 expression were significantly upregulated on DCs after 16 h of culturing the cells (Fig. 1E); the p values for upregulation were p < 0.01 for CD80 and p < 0.001 for CD86 (two-tailed paired t test, Fig. 1F).

Expression of receptors for IL-7 and TSLP on DCs

As IL-7Rα is a component of the receptors for both IL-7 and TSLP (Fig. 1A), the induction of responsiveness to these cytokines might reflect an upregulation of IL-7Rα expression on cultured DCs. As reported (17), we found that the great majority of ex vivo splenic DCs had no detectable IL-7Rα because isotype control and mAb against IL-7Rα stained the same amount of CD11cbright/MHC IIbright cells (Supplemental Fig. 2). Overnight culture of splenocytes resulted in no change in the binding of the isotype control Ab to CD11cbright/MHC IIbright cells (Supplemental Fig. 2), whereas anti–IL-7Rα staining showed a dramatic upregulation of this receptor chain (Fig. 2A). Abs to γc and to TSLPR stained essentially all freshly isolated DCs; o/n culturing resulted in only a modest enhancement in the intensity of staining for each receptor chain. Quantitatively, ∼1% of the total ex vivo DCs expressed IL-7Rα whereas ∼20% of the cells were positive for IL-7Rα after o/n culture (Fig. 2B). The upregulation of IL-7Rα on DCs was statistically highly significant (p < 0.001, two-tailed paired t test).

FIGURE 2.
FIGURE 2.

The increased responsiveness of splenic DCs to TSLP and IL-7 is due to robust increase in IL-7Rα expression upon o/n culture. (A) IL-7Rα, γc, and TSLPR expression of splenic DCs (CD11cbright/MHC IIbright cells) was measured either directly after harvesting the spleens (ex vivo) or after o/n culture. A representative example is shown. (B) Statistical analysis of five to eight independent experiments. Bars represent the average percentage of receptor chain–positive cells (±SEM indicated). ***p < 0.001, two-tailed, paired t tests. (C) TSLPR Ab specificity was tested using splenic CD4 T cells from wild-type or TSLPR−/− mice. The experiment was done twice (one wild-type and one TSLPR−/− mouse per experiment). (D) The specificity of IL-7Rα and γc Abs was tested on splenic CD4 and B220 cells, as indicated with Ab-specific isotype controls (green and red lines). The staining was done twice with two different wild-type B6 mice.

We confirmed the specificity of the polyclonal anti-TSLPR Ab by using various amounts of anti-TSLPR Ab or control Ig on cell type known to express TSLPR (splenic CD4 T cells) from wild-type and TSLPR-deficient mice (Fig. 2C). To test the specificity of IL-7Rα and γc Abs, we compared their staining profiles on B220+ cells and CD4 T cells. As expected, anti–IL-7Rα showed strong staining on CD4 T cells, but no staining on B cells, that are known to lack surface IL-7Rα, whereas anti-γc staining was positive on both cell types (Fig. 2D).

Induction of IL-7Rα in different splenic DC subsets

The CD11cbright/MHC IIbright splenic DCs are actually a group of subtypes characterized by differential expression of surface markers. Differential expression of CD4 and CD8 has been used to classify CD11cbright DCs as CD4+/CD8−, CD4−/CD8+, or CD4−/CD8− subclasses (26). The different subclasses of splenic DCs might have a different capacity to upregulate IL-7Rα. We stained splenic cells for IL-7Rα immediately ex vivo or after o/n culture as in Fig. 2, but we analyzed the expression of CD4 and CD8 simultaneously (26). We identified three major populations among the cells in the CD11c+/MHC II+ gate based on CD4 and CD8 expression (Fig. 3A). None expressed detectable IL-7Rα when tested immediately after harvest. Strikingly, the upregulation of IL-7Rα in o/n-cultured cells was most prominent on CD8− DC populations (Fig. 3B). Whereas o/n-cultured CD8+ DCs showed levels of IL-7Rα that were modestly changed when compared with CD8+ DCs ex vivo (<10% of the cells became IL-7Rα+), ∼25% of CD4+ DCs became positive for IL-7Rα (Fig. 3C). CD4−/CD8− DCs fell between CD4+ and CD8+ DCs; <20% of the cells became IL-7Rα+ during o/n culture. Quantitatively, the difference of IL-7Rα expression on CD8+ DCs was significant ex vivo and after o/n culture (p < 0.01 and p < 0.001, respectively, Fig. 3C).

FIGURE 3.
FIGURE 3.

IL-7Rα upregulation occurs mainly in CD8− splenic DCs. (A) Gating the o/n-cultured splenic DCs on the basis of CD4 and CD8 expression. Numbers indicate percentage of cells of total CD11c/MHC II–positive cells. (B) IL-7Rα expression was studied in different splenic DC subpopulations ex vivo or after o/n culture. A representative result from one experiment is shown. (C) Statistical analysis of IL-7Rα expression on splenic DC subpopulations from three independent experiments. The bars indicate the percentage of IL-7Rα+ cells in the indicated subpopulation (the means ± SEM are indicated). **p < 0.01, ***p < 0.001, two-tailed, paired t tests.

IL-4 and LPS enhance IL-7Rα expression in cultured DCs

Next, we studied how various soluble factors might regulate the IL-7Rα expression in cultured DCs. In T cells, IL-7 itself downregulates IL-7Rα transcription and IL-4 has also been associated in downregulation of IL-7Rα (27). Additionally, LPS is a potent activator of DCs via the TLR4–MyD88–NF-κB pathway and could control the cytokine sensitivity by regulating the expression of cytokine receptors on DCs. For these experiments, the cells were left o/n in either 10% serum containing culture medium alone, or with IL-4 (20 ng/ml), IL-7 (40 ng/ml), LPS (1 μg/ml), or IL-4 plus LPS, and then measuring IL-7Rα in CD11cbright, MHC IIbright cells, which were negative for CD3, B220, and F4/80 (Fig. 4A).

FIGURE 4.
FIGURE 4.

IL-4 induces IL-7Rα expression and TSLP responsiveness of splenic DCs. (A) Comparison of splenic DC populations after different o/n culturing conditions. (B and C) Statistical analysis of IL-7Rα (B) and CD80 (C) expression on splenic DCs. Bars indicate mean fluorescence intensity (MFI; with ±SEM). IL-7Rα and CD80 staining data are from three independent experiments (three animals used in each experiment). (D) STAT5 phosphorylation in splenic DCs (either ex vivo or o/n cultured with indicated stimulations) in response to either vehicle (PBS) or IL-7 or TSLP. The bars indicate normalized MFI of pSTAT5 (o/n-cultured unstimulated sample was given the value 1). Data are from three independent experiments (three animals used in each experiment). (E) IL-7Rα upregulation in different DC subpopulations in response to IL-4 was measured. MFI (±SEM) is indicated. *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed, paired t test was used. FSC, forward scatter; SSC, side scatter.

In splenic DCs, o/n culturing alone increased IL-7Rα expression significantly as compared with ex vivo–purified DCs (p = 0.0003). Strikingly, as compared with o/n alone–cultured DCs, adding IL-7 into the culture appeared to strongly downregulate IL-7Rα expression in DCs (p = 0.019), whereas adding IL-4 significantly increased IL-7Rα expression (p = 0.0001, Fig. 4B). LPS alone also induced IL-7Rα expression significantly, when compared with DCs cultured in medium alone (p = 0.0076), whereas adding IL-4 to LPS did not further increase the expression of IL-7Rα in DCs. The induction of IL-7Rα was not associated with further activation of DCs as judged by CD80 expression. CD80 was upregulated in o/n-cultured cells, but both IL-7 and LPS further induced CD80 expression, whereas IL-4 did not (Fig. 4C).

In line with changes observed in the expression of IL-7Rα, the induction of intracellular pSTAT5 by 15 min stimulation with TSLP was enhanced in splenic DCs that had been cultured in IL-4 when compared with DCs cultured in culture medium alone (p = 0.0037, Fig. 4D). Strikingly, no difference in TSLP responsiveness was observed between DCs that been cultured either in medium alone or in medium containing IL-7 (p = 0.5008), whereas IL-7 responses were slightly, but not statistically, significantly lower in IL-7–cultured cells when compared with medium alone (p = 0.0998). Albeit LPS upregulated IL-7Rα in splenic DCs (Fig. 4B), culturing these cells in LPS did not enhance their cytokine responsiveness (Fig. 4D). Taken together, TSLP-induced STAT5 phosphorylation was significantly enhanced only in DCs cultured in IL-4. The slight decrease in pSTAT5 responsiveness to IL-7 by DCs cultured in IL-7 was not due to decreased expression of γc by IL-7 stimulation (data not shown).

In DCs purified from lymph nodes (Supplemental Fig. 3), o/n culture significantly increased expression of IL-7Rα (p = 0.0010), whereas IL-4 appeared to enhance IL-7Rα, but this difference was not statistically significant (p = 0.0871), whereas IL-7 clearly downregulated IL-7Rα (p = 0.0118) in these cells (Supplemental Fig. 3). For CD80 expression in lymph node DCs, IL-7 induced CD80 expression whereas IL-4 had no effect on CD80 expression (Supplemental Fig. 3).

Human DCs upregulate IL-7Rα during o/n culture

In human peripheral blood myeloid DCs, not only IL-7Rα but also TSLPR are upregulated during o/n culture (19), whereas in human airway mucosal myeloid DCs TSLPR is constitutively activated (28). This could suggest differential regulation of the TSLP receptor system between two anatomic locations. To learn whether the observed IL-7Rα upregulation we discovered in murine DCs would occur in human DCs and would be linked particularly to stimulation with IL-4, we studied human peripheral blood DCs from healthy donors (n = 5) that were enriched as described in Materials and Methods. We analyzed HLA-DRbright/CD11cbright cells either directly ex vivo or after o/n culture on tissue culture plates (Fig. 5A). For o/n culture, we either left the cells unstimulated or stimulated the cells with human IL-4 or human TSLP. Quite differently from murine DCs, CD80 expression in human DCs was only modestly upregulated during o/n culture, but IL-4 and particularly TSLP strongly upregulated CD80 expression (Fig. 5B). For IL-7Rα expression, low expression ex vivo was strongly upregulated during o/n culture (p = 0.0012) and the upregulation was biphasic, indicating that IL-7Rα upregulation was not a feature of all HLA-DRbright/CD11cbright cells. Alternatively, only a portion of the cells was able to overcome possible inhibitory factors in the o/n cultures that were keeping IL-7Rα expression low. IL-4 stimulation itself did not induce IL-7Rα expression in human DCs (for o/n culture versus o/n culture with IL-4, p = 0.0933) whereas combining IL-4 stimulation to TSLP strikingly downregulated IL-7Rα expression (p = 0.0024) in human DCs. Thus, for human total HLA-DRbright/CD11cbright DCs, IL-4 did not upregulate IL-7Rα. It will be of interest to characterize the DC subpopulations that clearly upregulate IL-7Rα and determine whether IL-4 specifically regulates IL-7/TSLP sensitivity in these subpopulations.

FIGURE 5.
FIGURE 5.

IL-7Rα expression is upregulated in human peripheral blood DCs during o/n culture. (A) Gating strategy of enriched human peripheral DCs ex vivo (upper panel) or after o/n culture (lower panel). (B) A representative experiment of expression of IL-7Rα (left panel) or CD80 in human DCs from one healthy donor after indicated culture conditions. (C) Statistical analysis of IL-7Rα and CD80 expression in peripheral blood DCs from five healthy donors after indicated culture conditions/stimulations. The means ± SEM are indicated. **p < 0.01, ***p < 0.001, two-tailed, paired t tests. FSC, forward scatter; SSC, side scatter.

IL-4 increases IL-7Rα expression on mouse splenic DCs in vivo

The notion that IL-4 appeared to further upregulate IL-7Rα on o/n-cultured DCs in mice prompted us to ask whether IL-4 might have the same effect on DCs in vivo. Because the half-life of IL-4 in biologic fluids is rather short, we used the method described by Morris et al. (22), where complexing IL-4 to a soluble anti–IL-4 Ab prolongs the cytokine t1/2. Wild-type B6 mice (n = 5 per treatment) were either injected with anti–IL-4 or anti–IL-4 complexed to IL-4 (complex). To verify that the complex administration resulted in IL-4–mediated responses, we measured IL-7Rα and IL-4Rα on T cells. As expected (27), the expression of IL-7Rα was decreased in CD4 T cells in response to the complex (p < 0.0001), whereas the complex significantly upregulated IL-4Rα (p = 0.0092) in these cells (Fig. 6A, gating strategy for T cells is shown in Supplemental Fig. 4). For CD8 T cells, we found that the results were similar; IL-4 downregulated IL-7Rα (p = 0.0013) and upregulated IL-4Rα (p < 0.0001) expression (Fig. 6B). Because IL-4 upregulated IL-7Rα on DCs in vitro (Fig. 4B), we next asked whether the same occurs in vivo. As we measured IL-7Rα expression from mice treated with anti–IL-4, no effect on IL-7Rα expression was observed. However, for mice treated with complex we noticed consistent and statistically significant induction in IL-7Rα on DCs (Fig. 6C, left panel, p = 0.0005). In line with observations from in vitro cultures, CD80 was not induced by IL-4 in vivo (Fig. 6C, right panel).

FIGURE 6.
FIGURE 6.

IL-4 affects surface expression of IL-7Rα in T cells and DCs differently in vivo. Anti–IL-4 Ab alone (aIL-4) or mixed with IL-4 (complex) were administrated i.p. to B6 mice (five per group) twice 8 h apart. Sixteen hours later, mice were euthanized and splenocytes were harvested and analyzed based on CD3+/CD4+/CD8+ (A, B, and D) or CD11c+/MHC II+ (C) expression. (A) IL-7Rα (left panel) and IL-4Rα (right panel) expression in CD4 T cells. In all experiments dot represents expression level in one mouse (±SEM indicated). (B) IL-7Rα (left panel) and IL-4Rα (right panel) expression in CD8 T cells. (C) Expression of IL-7Rα (left panel) and CD80 (right panel) in splenic DCs. (D) Comparison of IL-4Rα expression in CD4 (left panel) and CD8 (right panel) T cells of untreated, aIL-4– and complex-treated B6 mice. **p < 0.01, *** p < 0.001 two-tailed, paired t test.

To confirm that anti–IL-4 was an appropriate control for these experiments, we compared IL-4Rα expression in CD4 and CD8 T cells. We found that IL-4Rα expression in either cell type was similar in untreated or anti–IL-4-treated mice, whereas complex-treated mice showed dramatic upregulation of this receptor chain (Fig. 6D).

Discussion

The requirement of IL-7Rα for both IL-7- and TSLP-induced signaling coupled with the distinct anatomical distribution of the two cytokines prompted us to study how they act on DCs because these cells have been implicated as direct targets of TSLP. In accordance with previous studies, we found that only a small minority of freshly isolated splenic or lymph node DCs expressed IL-7Rα (17, 19) and that the great majority of these cells failed to phosphorylate STAT5 in response to either TSLP or IL-7. Overnight culture of these cells, which is known to activate DCs (25), resulted in upregulation of IL-7Rα and rendered a substantial proportion of the cells responsive to both IL-7 and TSLP. Evidence has been presented indicating that TSLP can cause skin or mucosal DCs to acquire Th2-inducing potential. The mechanism by which this occurs is still uncertain, but one possibility, at least in the human DCs, is the TSLP-mediated upregulation of OX40L expression (29).

The failure of freshly isolated splenic DCs to express IL-7Rα and to show responsiveness to TSLP implies that if TSLP truly plays a major physiologic role in Th2 differentiation through its effects on skin and/or mucosal DCs, then a dual induction process is required—the induction of TSLP synthesis/secretion by skin/mucosal epithelial cells and the induction of TSLP responsiveness by the local DCs. Induction of TSLP expression has been studied to some degree. Among the stimulants known to cause induction of TSLP are vitamin D3, TNF-α, IL-4, and IL-13 (12, 13). Less is known about the mechanism through which IL-7Rα is induced on tissue DCs. Our study of splenic DCs implies that IL-4 and the LPS pathways regulate its in vitro induction. The notion that IL-7 appeared to inhibit IL-7Rα expression on DCs is interesting but it may also be a technical artifact. It is feasible that IL-7 in the culture media blocks the binding of the Ab recognizing IL-7Rα. This conclusion is supported by the fact that o/n IL-7 stimulation of DCs has no inhibitory effect on TSLP-induced STAT5 phosphorylation (Fig. 4C), indicating that the building blocks of the functional receptor for TSLP are available on the cell surface. The notion that addition of IL-4 into the o/n DC culture induced IL-7Rα expression but only modestly enhanced the responsiveness of the splenic DCs to IL-7 might be explained by the fact that IL-4 in the culture medium could keep the γc chain constantly occupied to the type I IL-4 receptor, thus limiting the increase in IL-7 signaling. Related to receptor bioavailability, increased expression of IL-7Rα on DCs could have another important consequence, namely reducing the bioavailability of IL-7 in biologic fluids, which plays a major role in survival of T cells (27).

Our studies imply that a coordinated induction of TSLP and IL-7Rα is essential for TSLP-mediated activation/differentiation of DCs. Note that recent work indicated that physiologically TSLP executes some of its functions via nonhematopoietic cells (30). This calls for further studies of TSLP signaling and the possibility that an alternative receptor may still exist because IL-7Rα is considered to be expressed solely by hematopoietic cells (31). Furthermore, comparison of murine B cells from WT or TSLPR−/− mice indicated that WT B cells express substantial amount of TSLPR, whereas they are negative for IL-7Rα. Would wild-type B cells from IL-7Rα–deficient mice respond to TSLP, and would this occur via Stat5 or independently of Stat5? Comparing genome-wide transcription analysis combined with cellular phosphorylation analysis in wild-type and IL-7Rα–deficient mice would clarify whether TSLP truly could signal without IL-7Rα.

The notion that IL-4, which itself induces TSLP production (14), also regulates a critical component of TSLP receptor complex on DCs in our experiments is interesting. A possibility remains that in the early induction phase of allergic inflammation, basophil-derived IL-4 (32) acts locally in barrier tissues on both keratinocytes to produce TSLP and simultaneously on DCs to upregulate the receptor for TSLP and, thus, IL-4 could orchestrate locally both production and responsiveness to TSLP on specific cell types. Further studies on functional characteristics of the IL-4/TSLP cytokine axis on IL-4–deficient mice will reveal how TSLP signaling and receptor expression (particularly IL-7Rα) are regulated by IL-4. Experiments following IL-7Rα expression on DCs utilizing mice genetically modified to lack the expression of IL-4, IL-4Rα, insulin receptor substrate 2, and Stat6 should answer the question of how IL-4 signaling regulates the expression of IL-7Rα on DCs.

Our observation that CD8+/CD4− splenic DCs showed less induction of IL-7Rα as compared with CD8−/CD4+ or CD8−/CD4− DCs implies that professional IL-12–producing DCs respond poorly to TSLP and IL-7 or that TSLP stimulation may divert the cells from acquiring or maintaining IL-12 producing capacity, at least in this experimental setting. As CD8+/CD4− DCs are specific producers of IL-12, the failure of these cells to acquire IL-7Rα expression would be rational.

The benefit of the local regulation of IL-7Rα could be explained by locally regulated TSLP production depending on the signals that alveolar, bronchial, and skin epithelial cells receive. Whereas migratory DCs in lymph nodes and their skin counterparts (17) do express IL-7Rα at low levels, possibly allowing some cytokine signaling, the induction of IL-7Rα (and ensuing cytokine responsiveness) we observed in this study is quite drastic. The notion that in human freshly isolated DCs both TSLPR and IL-7Rα are expressed at low levels (19) indicates that human and mouse DCs may differ in the steady-state expression of TSLPR. Alternatively, TSLPR expression in human DCs may be regulated by infectious and genetic background differences that are absent in inbred mouse lines housed in standardized animal facilities. We found that in human peripheral blood DCs from small group of individuals (n = 5), the expression of IL-7Rα was induced during o/n culture as expected (19), but the additive IL-4–mediated effect seen in mice was not observed. One explanation could be that human DCs are continuously encountering Ags and are thus more activated than inbred mouse DCs from sterile housing. This idea is supported by the finding that CD80 expression was only modestly upregulated in human peripheral blood DCs as compared with splenic murine DCs. Thus, the phenotype of resting human DCs is quite different from corresponding mouse cells. Another point to consider is the fact that myeloid human DCs from different anatomical locations differ in their expression of TSLPR (19, 28), which could be explained by the fact that TSLP is expressed closer to epithelial and mucosal barriers and thus “sensing” of the cytokine is restricted to the cells closer these structures. Also, note that both IL-7Rα and TSLPR expression on DCs could be under active inhibitory regulation, particularly while in circulation. In murine CD4 T cells, IL-7Rα is downregulated by various cytokines (27), which could be the case for human DCs too. If this were the case, peripheral blood DCs would inherently express low levels of one or both of these receptor chains and only upon appropriate activation signal induce receptor chain expression, resulting in increased responsiveness to cytokine (TSLP/IL-7). Such a system would protect DCs against inappropriate activation by type 2–polarizing TSLP in the peripheral blood. Future work characterizing anatomy and activation status of various human DC populations in the context of TSLP (and IL-7) responses as well as expression of TSLPR and IL-7Rα will be a fruitful endeavor.

Another point to be considered is how much of the action of TSLP in Th2 responses is on CD4 T cells. Although there is the possibility that the STAT5 activation known to be critical to in vitro Th2 differentiation is mediated by TSLP in vivo, TSLP seems an unlikely cytokine to mediate its function in secondary lymphoid organs because its principal sites of production appear to be at mucosal and skin surfaces. If STAT5 activation is as essential for in vivo Th2 differentiation as it is for in vitro differentiation, IL-2 or IL-7 would appear to be far more likely to mediate this function during the initial phases of T cell priming. However, once activated CD4 T cells migrate to the tissues, TSLP-induced STAT5 phosphorylation could be important in completing or sustaining Th2 differentiation and/or survival.

Overall, our results provide a mechanistic explanation how DC responsiveness to TSLP in mouse can be modified simply by induction of IL-7Rα, the receptor chain required for both IL-7 and TSLP responses.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

Animal facilities both at National Institute of Allergy and Infectious Diseases and at School of Medicine, University of Tampere are thanked for support in animal experimentation.

Footnotes

  • This work was supported by the National Institute of Allergy and Infectious Diseases Intramural Research Program (to X.C., S.C., R.Y., W.E.P., J.Z., and I.S.J.), the Finnish Medical Foundation (to I.S.J.), the Sigrid Juselius Foundation (to I.S.J. and M.P.), the Tampere Tuberculosis Foundation (to I.S.J. and M.P.), Competitive State Research Financing of the Expert Responsibility Area of Tampere University Hospital Grants 9M080, 9N056, and 9S051 (to M.P.), Fimlab Laboratories Grant X51409 (to I.S.J.), Academy of Finland Projects 263955 and 135980 (to M.P.), and by the Emil Aaltonen Foundation (to L.K. and M.P.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    γc
    common γ chain
    DC
    dendritic cell
    MHC II
    MHC class II
    o/n
    overnight
    TSLP
    thymic stromal lymphopoietin
    TSLPR
    TSLP receptor.

  • Received April 27, 2016.
  • Accepted March 13, 2017.

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