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. 2014 Aug 14;15(1):90.
doi: 10.1186/s12931-014-0090-5.

Adjuvant-dependent regulation of interleukin-17 expressing γδ T cells and inhibition of Th2 responses in allergic airways disease

Emily M Nakada, Jichuan Shan, Margaret W Kinyanjui, Elizabeth D Fixman

Adjuvant-dependent regulation of interleukin-17 expressing γδ T cells and inhibition of Th2 responses in allergic airways disease

Emily M Nakada et al. Respir Res. .

Abstract

Background: Th2 immune responses are linked primarily to mild and moderate asthma, while Th17 cells, Interleukin-17A (IL-17) and neutrophilia have been implicated in more severe forms of disease. How Th2-dependent allergic reactions are influenced by Th17 and IL-17-γδ T cells is poorly understood. In murine models, under some conditions, IL-17 promotes Th2-biased airway inflammatory responses. However, IL-17-γδ T cells have been implicated in the inhibition and resolution of allergic airway inflammation and hyperresponsiveness (AHR).

Methods: We compared airway responses in Balb/c mice sensitized to OVA with (and without) a Th2-skewing aluminum-based adjuvant and the IL-17 skewing, complete Freund's adjuvant (CFA). AHR was measured invasively by flexiVent, while serum OVA-IgE was quantified by an enzyme immunoassay. Airway inflammatory and cytokine profiles, and cellular sources of IL-17 were assessed from bronchoalveolar lavage and/or lungs. The role of γδ T cells in these responses was addressed in OVA/CFA sensitized mice using a γδ T cell antibody.

Results: Following OVA challenge, all mice exhibited mixed eosinophilic/neutrophilic airway inflammatory profiles and elevated serum OVA-IgE. Whereas OVA/alum sensitized mice had moderate inflammation and AHR, OVA/CFA sensitized mice had significantly greater inflammation but lacked AHR. This correlated with a shift in IL-17 production from CD4+ to γδ T cells. Additionally, OVA/CFA sensitized mice, given a γδ TCR stimulatory antibody, showed increased frequencies of IL-17-γδ T cells and diminished airway reactivity and eosinophilia.

Conclusions: Thus, the conditions of antigen sensitization influence the profile of cells that produce IL-17, the balance of which may then modulate the airway inflammatory responses, including AHR. The possibility for IL-17-γδ T cells to reduce AHR and robust eosinophilic inflammation provides evidence that therapeutic approaches focused on stimulating and increasing airway IL-17-γδ T cells may be an effective alternative in treating steroid resistant, severe asthma.

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Figures

Figure 1
Figure 1
Serum OVA-specific IgE and airway inflammatory responses are enhanced in OVA/CFA sensitized mice. BALB/c mice were IP OVA sensitized without adjuvant (OVA/sal), or in the presence of alum (OVA/alum) or CFA (OVA/CFA). Corresponding control groups were injected with saline, alum or CFA. (A) Serum OVA-IgE levels. OVA-IgE was undetectable in control mice. Mean (+SEM) from 8–12 mice per group from a minimum of 3 independent experiments. One-way ANOVA, Holm-Sidak. (B) BAL fluid differential cell counts from OVA sensitized groups (solid bars) and their respective adjuvant controls (striped bars). Mean total cells, macrophages, eosinophils, neutrophils and lymphocytes (+SEM) are shown for 11–16 total mice from at least 3 independent experiments. Two-way ANOVA, Holm-Sidak. (C) Relative expression of CCL2, CCL11, CCL24, CXCL1 and CXCL5 in the lung following OVA challenge assessed by real-time PCR. Expression is presented as fold increase relative to the saline control. Results represent the means (+SEM) from 5–6 total mice per group from a minimum of 2 independent experiments. Two-way ANOVA, Holm-Sidak. (A-C) §p < 0.05 OVA sensitized groups vs. respective controls (antigen-dependent effects), *p < 0.05 comparisons between OVA groups (adjuvant-dependent effects).
Figure 2
Figure 2
AHR is absent in OVA/CFA sensitized mice and Th1/Th2 BAL fluid cytokine levels are influenced by the adjuvant used during OVA sensitization. Total lung resistance and elastance were assessed 24 h after the last airway OVA challenge. Mean (±SEM) respiratory system (A) resistance and (B) elastance to increasing concentrations of methacholine (left panels) for the 3 OVA-sensitized mouse groups, as well as at the 50 mg/ml concentration (right panels) for all 6 groups. Control groups were removed from left panel figures for clarity. Data from 8–14 total mice per group from 2 independent experiments. (C) Mean BAL fluid levels (+SEM) of IL-4, IL-5, IL-13 & IL-17 were assessed from 7–12 total mice per group, from at least 2 independent experiments. (A-C) Two-way ANOVA, Holm-Sidak. §p < 0.05 OVA sensitized groups versus respective controls (antigen-dependent effects); *p < 0.05 comparisons within the OVA sensitized groups (adjuvant-dependent effects), specifically #p < 0.05 OVA/alum vs. OVA/CFA, %p < 0.05 OVA/alum vs. OVA/sal.
Figure 3
Figure 3
IL-17 expressing cells are present in the BAL fluid of all OVA sensitized and challenged mice. Cells were stimulated with PMA/ionomycin and stained to detect IL-17. (A) Representative flow cytometry plots are shown. (B) The frequencies of IL-17 expressing cells for the OVA groups are shown. One-way ANOVA, Holm-Sidak. (C) The total number of IL-17 expressing cells are shown for OVA groups. Two-way ANOVA, Holm-Sidak. Mean values (+SEM) from 7–11 total mice per group, from 2 independent experiments. (B-C) *p < 0.05 comparisons between OVA groups.
Figure 4
Figure 4
OVA/CFA sensitized mice have more γδ + IL-17 + T cells in the BAL fluid. BAL fluid cells were stimulated with PMA/ionomycin and triple stained with α-CD4, α-γδ TCR and α-IL-17 antibodies. (A) Representative flow cytometry plots of cells in BAL fluid (top panel) and lung (bottom panel) gated first to identify IL-17+ cells and subsequently to identify frequencies of CD4 and γδ T cells. (B) The frequency distribution of IL-17+ cells within the CD4 and γδ T cell populations is presented as the ratio (γδ/CD4) of these cells. One-way ANOVA, Holm-Sidak. (C) Total numbers of CD4+IL-17+ and γδ+IL-17+ populations calculated from the frequency of these cells and the total cell counts are shown. Two-way ANOVA, Holm-Sidak. (D) The distribution of total γδ+IL-17+ and CD4+IL-17+ T cells is presented as the ratio (γδ/CD4) of these cells. One-way ANOVA, Holm Sidak. (B-D) Data are from 7–11 total mice per group from at least 2 independent experiments. *p < 0.05 comparisons between OVA groups (adjuvant-dependent effects), §p < 0.05 OVA sensitized groups versus respective controls (antigen-dependent effects).
Figure 5
Figure 5
Frequencies of total IL-17 expressing cells and of IL-17-γδ cells are increased in OVA/CFA sensitized mice treated with a γδ TCR stimulatory antibody. OVA/CFA sensitized mice were IV injected with a γδ TCR stimulatory antibody (UC7-13D5) or isotype control before airway challenge. BAL fluid cells were stimulated with PMA/ionomycin and stained with α-γδ TCR and α-IL-17 antibodies. (A) Representative flow cytometry plots of BAL fluid cells. The mean frequencies (left panels) and numbers (right panels) of (B) IL-17-γδ and (C) IL-17+ BAL fluid cells are presented for 5 total mice per group from 2 independent experiments. (B-C) Unpaired, two-tailed t-test. *p < 0.05.
Figure 6
Figure 6
OVA/CFA sensitized mice receiving a γδ TCR stimulatory antibody, have reduced airway eosinophilia and AHR. OVA/CFA sensitized mice were IV injected with a γδ TCR (UC7-13D5) stimulatory antibody or isotype control before airway challenge. The mean (A) total cell counts and (B) frequencies of macrophages, eosinophils, neutrophils and lymphocytes (+SEM) are shown for 5 total mice per group from 2 independent experiments. (C) Total lung resistance and elastance were assessed 24 h after the last airway OVA challenge. Mean (±SEM) respiratory system resistance and elastance to increasing concentrations of methacholine are shown. (A-C) Unpaired, two-tailed t-test. *p < 0.05, ns = not significant.
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