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. 2014 Jul;134(7):1903-1911.
doi: 10.1038/jid.2014.61. Epub 2014 Jan 31.

IL-9 regulates allergen-specific Th1 responses in allergic contact dermatitis

Juan Liu  1 , Erin Harberts  2 , Antonella Tammaro  3 , Nicholas Girardi  4 , Renata B Filler  5 , Rita Fishelevich  1 , Angela Temann  6 , Paula Licona-Limón  6 , Michael Girardi  5 , Richard A Flavell  6 , Anthony A Gaspari  7
Affiliations

IL-9 regulates allergen-specific Th1 responses in allergic contact dermatitis

Juan Liu et al. J Invest Dermatol. 2014 Jul.

Abstract

The cytokine IL-9, derived primarily from T-helper 9 (Th9) lymphocytes, promotes expansion of the Th2 subset and is implicated in the mechanisms of allergic asthma. We hypothesize that IL-9 also has a role in human allergic contact dermatitis (ACD). To investigate this hypothesis, skin biopsy specimens of positive patch-test sites from non-atopic patients were assayed using quantitative PCR and immunohistochemistry. The cytokines IFN-γ, IL-4, IL-17A, IL-9, and PU.1, a Th9 associated transcription factor, were elevated when compared with paired normal skin. Immunohistochemistry on ACD skin biopsies identified PU.1+ CD3+ and PU.1+ CD4+ cells, consistent with Th9 lymphocytes, in the inflammatory infiltrate. Peripheral blood mononuclear cells from nickel-allergic patients, but not nonallergic controls, show significant IL-9 production in response to nickel. Blocking studies with mAbs to HLA-DR (but not HLA-A, -B, -C) or chloroquine significantly reduced this nickel-specific IL-9 production. In addition, blockade of IL-9 or IL-4 enhanced allergen-specific IFN-γ production. A contact hypersensitivity model using IL-9(-/-) mice shows enhanced Th1 lymphocyte immune responses, when compared with wild-type mice, consistent with our human in vitro data. This study demonstrates that IL-9, through its direct effects on Th1 and ability to promote IL-4 secretion, has a regulatory role for Th1 lymphocytes in ACD.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

Figure 1
Figure 1. Increased expression of IL-9 and associated genes in AC and the detection of Th9 cells in allergic contact dermatitis
(A) Mean relative-gene expression of IL-9, was elevated on average 5 (range 2–18) fold higher than paired control skin. IL-9 increase is similar to increases in IFN-γ, IL-4, IL-17A, CCL11 and CD3ε. (B) Mean relative gene expression of IL-9/Th9-associated transcription factors PU.1, IRF4, ETS1 and GcN5 was 2–3 (range 1.5–8) fold greater than paired control skin. Sections of skin biopsy specimens from a representative (C) Stained slides from an ACD patient were stained with anti-PU.1 (red) and anti-CD4 (green); nuclei are counter-stained with DAPI (blue). Stained T lymphocytes were identified as (D) PU.1+/CD3+ or (E) PU.1+/CD4+. No PU.1+/CD8+ cells were identified. To quantify cell populations, twelve fields from each double-stained section were counted with a mean of 40 infiltrating cells per field. Mean +/− SEM is depicted. For gene expression studies, skin biopsy samples were taken from positive patch tests to nickel or cobalt from seven different patients (ACD 6–12 in Table 1). For the immunochemistry, skin biopsy specimens were taken from five different positive patch tests in five different patients (ACD1–5 in Table 1).
Figure 2
Figure 2. Allergen-specific IL-9 production by PBMC from nickel-allergic patients
PBMC were isolated from 7 different patients with ACD to nickel (Table 1), and cultured with nickel chloride (1–10 μg/ml of culture medium) for ninety six hours. Supernatants were harvested, and the following cytokines were assayed in cell-free culture supernatants: IL-9, IFN-γ, IL-4, and IL-2 (data depicted represent mean +/− SD of samples assayed in triplicate). (A) Dose response curve to Nickel titration from two nickel allergic patients and two nickel tolerant patients. (B) IL-9 production from the same two nickel allergic and tolerant patients, demonstrating that both groups can produce IL-9 in an equivalent manner after PHA stimulation. (C) Scatter plot represents the peak cytokine production from the seven allergic patients. The scatter plot of peak cytokine production (horizontal bar represents mean value) for both IFN-γ and IL-9 were significantly higher than of IL-4 (P<0.001), with the average peak IL-9 production is similar to IFN-γ (P>0.05).
Figure 3
Figure 3. IL-9 production depends on exogenous antigen presentation to Th9-lymphocytes
IL-9 production was measured 96h after stimulation of PBMC with medium containing nickel chloride (10μg/ml). Samples are incubated with either media, anti-HLA-DR monoclonal antibody (0.25μg/ml and 1μg/ml), anti-HLA-A,B,C monoclonal antibodies (0.25μg/ml and 1μg/ml), or chloroquine (5μM and 20μM) 2 hours prior to stimulation with nickel. Antibodies to HLA-A,B, C had little effect on IL-9 production, while Abs to HLA-DR or incubation with chloroquine resulted in significantly reduced IL-9 production. Mean +/− SD of samples assayed in triplicate is depicted. Our results suggest the processing of nickel via the exogenous pathway of antigen presentation is necessary for PBMC production of IL-9. Experiment was repeated with PBMC from three different patients, and found to be reproducible.
Figure 4
Figure 4. IL-4 but not IL-9 directly regulates IFN-γ during allergen-specific Th1 activation in vitro
(A) PBMC from a nickel allergic patient were cultured in medium alone or in medium containing nickel-chloride, the indicated doses of IL-9 alone, IL-4 alone, or IL-9 and IL-4 together were added to the nickel-chloride stimulated PBMC. Supernatants were collected 96 h after nickel-stimulation. (B) IL-4 secretion was also measured after addition of IL-9. (C) IL-9 was measured in those culture conditions which had added IL-4. (D) Specific blocking anti-sera were added to the nickel culture, and IFN-γ was assayed using multiplex ELISA. Data depicted represent mean +/− SD of samples assayed in triplicate. This experiment was repeated with PBMC three times, and found to be reproducible. The data in (A–D) represent PBMC from distinct nickel-allergic patients.
Figure 5
Figure 5. The regulatory role of IL-9 in mouse contact hypersensitivity
(A) Ear swelling response of WT or IL-9−/− mice (n=5/group) was measured over time. (B) Ear punches taken 24 hours after elicitation are stained with H&E; depicted ears from WT (left) and IL-9−/− mice (right). (C) Gene expression was measured by qPCR from ear skin 24 hours after elicitation. Mean +/− SD fold-change in gene expression is shown (n=4 mice/group, *,p<0.05). (D) Sera (n=5 mice/group) from WT or IL-9−/− mice 21 days after sensitization, was measured for DNP-specific IgG1 or (E) IgG2a by ELISA. Levels of DNP specific Ab from pre-bleed sera (prior to sensitization) was subtracted to calculate the net DNP-specific response. Data depicted represent mean +/− SD (***p<0.001). (E) Irritant contact dermatitis to croton oil in WT and IL-9−/− mice; data indicate mean +/− SD (p<0.0023).
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