Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 193 (6), 2631-40

Th17 Cells Demonstrate Stable Cytokine Production in a Proallergic Environment

Affiliations

Th17 Cells Demonstrate Stable Cytokine Production in a Proallergic Environment

Nicole L Glosson-Byers et al. J Immunol.

Abstract

Th17 cells are critical for the clearance of extracellular bacteria and fungi, but also contribute to the pathology of autoimmune diseases and allergic inflammation. After exposure to an appropriate cytokine environment, Th17 cells can acquire a Th1-like phenotype, but less is known about their ability to adopt Th2 and Th9 effector programs. To explore this in more detail, we used an IL-17F lineage tracer mouse strain that allows tracking of cells that formerly expressed IL-17F. In vitro-derived Th17 cells adopted signature cytokine and transcription factor expression when cultured under Th1-, Th2-, or Th9-polarizing conditions. In contrast, using two models of allergic airway disease, Th17 cells from the lungs of diseased mice did not adopt Th1, Th2, or Th9 effector programs, but remained stable IL-17 secretors. Although in vitro-derived Th17 cells expressed IL-4Rα, those induced in vivo during allergic airway disease did not, possibly rendering them unresponsive to IL-4-induced signals. However, in vitro-derived, Ag-specific Th17 cells transferred in vivo to OVA and aluminum hydroxide-sensitized mice also maintained IL-17 secretion and did not produce alternative cytokines upon subsequent OVA challenge. Thus, although Th17 cells can adopt new phenotypes in response to some inflammatory environments, our data suggest that in allergic inflammation, Th17 cells are comparatively stable and retain the potential to produce IL-17. This might reflect a cytokine environment that promotes Th17 stability, and allow a broader immune response at tissue barriers that are susceptible to allergic inflammation.

Figures

Figure 1
Figure 1
Generation of Il17fCost mice. A, Schematic of the Il17f locus, targeting vector and the targeted Il17f locus. B, Naïve CD4+ T cells were isolated from the spleen of Il17f+/+RsYFP/YFP, Il17f+/CostRsYFP/YFP or Il17fCost/CostRsYFP/YFP mice and stimulated under Th17-polarizing conditions for 5 days. Cells were re-stimulated with PMA and ionomycin and YFP and cytokine expression was analyzed by flow cytometry. Cells are gated on the live population (left and middle panels) and live YFP+ population (right panel). C, Naïve CD4+ T cells were isolated from the spleen of Il17f+/CostRsYFP/YFP mice and stimulated under T helper cell polarizing conditions for 5 days. Cells were re-stimulated with PMA and ionomycin and YFP and cytokine expression was analyzed by flow cytometry in the live population. D, EAE was induced in Il17f+/CostRsYFP/YFP mice. Mice were sacrificed 19 days after induction of disease and spleen and brain were harvested. Splenocytes (data not shown) and mononuclear cells from the brain were re-stimulated with PMA and ionomycin and cytokines were analyzed by flow cytometry in live total CD4+ (gate a), CD4+YFP+ (gate b) or CD4+YFP lymphocytes (gate c). Representative dot plots are shown and data are representative of 2 or more independent experiments with 3–4 mice per group (B–D).
Figure 2
Figure 2
Th17 cells display an altered cytokine profile when cultured under T helper cell-polarizing conditions. A–F, Naïve CD4+ T cells were isolated from the spleen of Il17f+/CostRsYFP/YFP mice and stimulated under Th17-polarizing conditions for 5 days (Round 1). Cells were re-stimulated and cultured under long-term Th17 polarizing conditions for another five days (Round 2). Live YFP+ cells were sorted by flow cytometry and cultured under Th1, Th2, Th9 or long-term Th17-polarizing conditions for five days (Round 3). A, After 2 rounds of stimulation under Th17-polarizing conditions, cells were re-stimulated with PMA and ionomycin and cytokines were analyzed by flow cytometry. Cells are gated on the live YFP+ population. Representative dot plots are shown. B, After culture under polarizing conditions (Round 3), cells were re-stimulated, stained and analyzed as in A. Cells are gated on the live YFP+ population. Representative dot plots are shown. C–F, Graphical representation of the data displayed in B. The data are the mean ± SEM of 5 mice. Statistical analysis in C–F was performed using the one-way ANOVA. *p < 0.05, compared with Th17 → Th17 samples. Data are representative of three or more independent experiments (A–F).
Figure 3
Figure 3
Expression of transcription factors in Th17 cells cultured under Th cell-polarizing conditions. A–C, Naïve CD4+ T cells were isolated from the spleen of Il17f+/CostRsYFP/YFP mice and stimulated under Th17-polarizing conditions for 5 days (Round 1). Cells were re-stimulated and cultured under long-term Th17-polarizing conditions for another five days (Round 2). Live YFP+ cells were sorted by flow cytometry and cultured under Th1, Th2, Th9 or long-term Th17-polarizing conditions for three days (Round 3) and live YFP+ cells were sorted for further analysis. A, Expression of the indicated transcription factors was assessed in unstimulated cells by flow cytometry. Representative histograms are shown. B, Graphical representation of the mean fluorescence intensity (MFI) data shown in A. The data are the mean ± SEM of 3 mice. C, RNA was isolated from sorted live YFP+ cells on the third day of round 2 (Th17 pre-switch) or sorted live YFP+ cells after three days of the third round of culture. Expression of the indicated genes was measured using quantitative PCR; samples were normalized to the expression of β2-microglobulin mRNA and are relative to Th17 → Th1 cells (Tbx21), Th17 → Th2 cells (Gata3) or Th17 → Th17 cells (Rorc, Batf, Irf4, Maf). The data are the mean ± SEM of 3 mice. Statistical analysis in B–C was performed using the one-way ANOVA. *p < 0.05, compared with Th17 → Th17 samples. Data are representative of 3 or more independent experiments (A–C).
Figure 4
Figure 4
Stability of Th17 cells in an acute model of AAD. A–C, Il17f+/CostRsYFP/YFP mice were sensitized i.p. with OVA and alum on days 0 and 7, challenged i.n. with PBS or OVA on days 14–19 and sacrificed 24 h after the final challenge. Cells were isolated from the BAL and lung (data not shown) for further analysis. A, Total cell number was calculated in the BAL of control (PBS) or allergic (AAD) mice (top panel). CD4+YFP+ cell number was determined from the BAL of control or allergic mice using flow cytometry (bottom panel). Data are the mean ± SEM of 3–5 mice. B, BAL cells were stimulated with PMA and ionomycin and cytokines were analyzed by flow cytometry in live CD4+ (left panel) or CD4+YFP+ lymphocytes (right panel). Representative dot plots are shown. C, Graphical representation of the CD4+YFP+ data displayed in B. The data are the mean ± SEM of 5 mice. Data are representative of 3 or more independent experiments (A–C).
Figure 5
Figure 5
Stability of Th17 cells in a chronic model of AAD. A–B, Il17f+/CostRsYFP/YFP mice received i.n. doses of HDM for 3 consecutive days each week for 5 weeks and were sacrificed 24 h after the final challenge. Cells were isolated from the BAL and lung (data not shown) for further analysis. A, The total cell number was calculated in the BAL of control (PBS) or allergic (AAD) mice (top panel). The CD4+YFP+ cell number was determined from the BAL of control or allergic mice using flow cytometry (bottom panel). Data are the mean ± SEM of 3–5 mice. B, BAL cells were stimulated with PMA and ionomycin and cytokines were analyzed by flow cytometry in live CD4+YFP+ lymphocytes. The data are the mean ± SEM of 3 mice.
Figure 6
Figure 6
Cytokine receptor expression from Th17 cells derived in vitro or in vivo during the development of AAD. A, For Th17 pre-switch cells, naïve CD4+ T cells were isolated from the spleen of Il17f+/CostRsYFP/YFP mice and stimulated under Th17-polarizing conditions for 5 days (Round 1). Cells were re-stimulated and cultured under long-term Th17-polarizing conditions for another five days (Round 2) and live YFP+ cells were sorted by flow cytometry. For other Th samples, naïve CD4+ T cells were isolated from the spleen of Il17f+/+RsYFP/YFP littermate control mice and stimulated under Th-polarizing conditions for five days before RNA was isolated. Expression of the indicated genes was measured using quantitative PCR; samples were normalized to the expression of β2-microglobulin mRNA and are relative to Th17 cells (Il23r), Th1 cells (Il12rb2) or Th2 cells (Il4ra). The data are the mean ± SEM of 3–5 mice. B, Th2 and Th17 pre-switch cells were cultured as in A and IL-4Rα expression was analyzed in live YFP and YFP+ populations by flow cytometry. Percentages indicated are of the respective YFP+ or YFP populations. Representative dot plots are shown. C, OVA and alum-induced AAD was induced in Il17f+/CostRsYFP/YFP mice as in Figure 4. Cells were isolated from the BAL and lung (data not shown) and IL-4Rα expression was analyzed in YFP and YFP+ CD4+ lymphocytes from control (PBS) or allergic (AAD) mice. The cells are gated on live CD4+ lymphocytes. Representative dot plots are shown with the mean ± SD of 2–4 mice displayed. Statistical analysis in A was performed using the one-way ANOVA. *p < 0.05, compared with Naïve samples. Data are representative of 2 independent experiments (A, C).
Figure 7
Figure 7
Stability of Ag-specific in vitro-derived Th17 cells upon adoptive transfer to allergic mice. A–D, WT mice were injected i.p. with PBS or sensitized i.p. with OVA and alum (O/A) on days 0 and 7. All mice were injected i.v. with sorted YFP+ Th17 cells (1×105) differentiated from Il17f+/CostRs+/YFP –OT-ll mice (2 rounds under Th17-polarizing conditions) on day 20. Mice were challenged i.n. with PBS or OVA on days 21–26 and sacrificed 24 h after the final challenge. Cells were isolated from the BAL and lung for further analysis. A, Total cell number was calculated in the BAL of each group of mice. Data are the mean ± SEM of 7–8 mice. B, Sorted YFP+ Th17 cells differentiated from Il17f+/CostRs+/YFP –OT-ll mice (2 rounds under Th17-polarizing conditions) were stimulated with PMA and ionomycin and cytokines were analyzed by flow cytometry (Pre-transfer YFP+). C, BAL (data not shown) and lung cells were stimulated with PMA and ionomycin and cytokines were analyzed by flow cytometry in live YFP (endogenous) or YFP+ (Post-transfer) CD4+ lymphocytes. Representative dot plots are shown. D, Graphical representation of the Pre-transfer YFP+ and Post-transfer CD4+YFP+ data displayed in B–C. Different IL-17A antibody clones were used in the top versus bottom two panels of dot plots in BC. The data are from pooled Th17 cells (Pre-transfer) or the mean ± SEM of 4 mice (Post-transfer).

Similar articles

See all similar articles

Cited by 8 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback