Increased expression of nuclear factor of activated T cells 1 drives IL-9-mediated allergic asthma.

To the Editor:

Nuclear factor of activated T cells (NFAT) is a family of transcription factors activated by dephosphorylation mediated by Ca++-activated calcineurin. NFAT coordinates different aspects of T-cell development and activation of T, B, natural killer, and mast cells and is the target of the immunosuppressive drug cyclosporin A. We reported recently that targeted deletion of NFATc1 in T cells resulted in inhibition of TH2 and TH17 differentiation.

Here we first investigated NFATc1 mRNA expression in PBMCs isolated from healthy and asthmatic children from the PreDicta cohort. Children with allergic asthma expressed significantly more NFATc1 mRNA than healthy control subjects (Fig 1, A-C, and Tables I and II). Furthermore, we found that asthmatic children with a positive skin test result had significantly increased expression of NFATc1 mRNA compared with healthy control subjects (Fig 1, D, and Tables I and II). These results suggested that NFATc1 might have a role in allergic asthma. Consistent with a TH2-inducing function of NFATc1 and a TH2 cytokine inhibitory property of the TH1 cytokine IFN-γ (IFNG), levels of this TH1 cytokine were found to be downregulated in children with asthma and a positive skin test result (Fig 1, E).

Fig 1

Increased expression of NFATc1 in atopic children with asthma. A,NFATc1 gene transcript isoforms or variants. B, Schematic representation of the PreDicta analysis. C,NFATc1 mRNA expression was analyzed by using quantitative real-time PCR in PBMCs (n = 12-17 children per group). D, Children shown in Fig 1, C, were further subdivided into healthy children with negative skin test results (n = 5) and asthmatic children with positive skin test results (n = 13). E,IFNG mRNA expression analyzed by using quantitative real-time PCR in RNA isolated from whole blood (n = 6 and 15 children per group). F, Schema of the subjects and their obtained gene expression profiles from Asthma BRIDGE Cohort. Statistical significance in this figure was evaluated with the Student t test. *P ≤ .05 and **P ≤ .01. Data are presented as means ± SEMs.

Table I

Clinical outcome of children with asthma participating in the WP1-UKER cohort in the European PreDicta study

Group/patient	Age (y)	Sex	PBMCs (106 cells/mL of blood)	Skin prick test	FEV1 actual value (%)	Asthma control	Asthma severity	Cigarette exposure/d	No. of symptomatic episodes before B0
Asthma
1	6	M	1.75	Positive	126	C	MOPA	0	0
2	6	M	2.27	Positive	80	PC	MOPA	0	3
3	5	F	2.05	Positive	108	PC	MIPA	25	—
4	6	M	1.98	Positive	128	C	MIPA	0	1
5	5	M	2.58	Positive	102	PC	I	0	4
6	5	F	2.62	Positive	129	C	I	0	1
7	5	M	2.32	Positive	143	PC	I	0	4
8	6	F	1.57	Positive	94	PC	I	0	3
9	4	M	1.96	Positive	115	PC	MOPA	0	3
10	5	F	1.93	Positive	92	U	MOPA	0	15
11	6	F	2.05	Positive	111	C	I	0	2
12	5	M	1.14	Positive	99	C	I	0	0
13	4	F	2.89	Negative	135	C	I	0	4
14	4	M	1.91	ND	96	C	I	5	4
15	5	M	1.29	Positive	80	C	I	0	2
16	5	M	1.83	Positive	86	C	I	0	1
17	5	M	2.50	Positive	107	C	I	0	2
18	4	M	1.54	Positive	71	PC	I	0	3
19	4	M	2.20	Negative	86	C	I	0	3
20	5	F	2.27	ND	98	C	I	0	1
21	5	F	1.77	Negative	—	U	MIPA	0	20
22	5	M	1.39	Positive	81	C	MIPA	0	3-5
Mean	5.00	14 M	1.99		103.19			1.36
SEM	0.14	8 F	0.09		4.20			1.15

B0, Baseline visit; C, controlled; F, female; I, intermittent; M, male; MIPA, mild persistent asthma; MOPA, moderate persistent asthma; ND, not done; PC, partially controlled; U, uncontrolled; UKER, Uniklinikum Erlangen; WP1, Work Program 1.

Table II

Clinical outcome of control children participating in the WP1-UKER cohort in the European PreDicta study

Group/patient	Age (y)	Sex	PBMCs (106 cells/mL of blood)	Skin prick test	FEV1 actual value (%)	Cigarette exposure/d	No. of symptomatic episodes before B0
Healthy subjects
1	6	M	1.14	ND	—	0	—
2	6	F	2.95	ND	121	0	—
3	5	F	1.79	Negative	81	0	—
4	4	M	2.01	ND	—	0	—
5	6	M	1.41	ND	105	0	—
6	4	F	2.14	ND	109	0	—
7	6	M	2.86	ND	87	0	—
8	4	M	1.73	Negative	100	0	—
9	5	F	1.67	ND	112	0	—
10	5	F	2.01	Positive	119	0	—
11	4	M	2.43	Positive	116	0	—
12	5	M	2.32	ND	111	0	—
13	4	M	2.40	Negative	109	0	—
14	4	F	1.60	Negative	109	0	—
15	5	M	2.22	Negative	92	0	—
16	4	M	2.88	Negative	—	0	—
17	4	M	1.38	Negative	109	0	—
18	5	M	1.13	ND	110	0	—
19	4	M	1.77	ND	118	0	—
20	5	F	1.50	ND	—	0	—
Mean	4.75	13 M	1.97		106.75	0
SEM	0.17	7 F	0.12		2.48	0

B0, Baseline visit; F, female; M, male; ND, not done; UKER, Uniklinikum Erlangen; WP1, Work Program 1.

As in PBMCs from the PreDicta cohort, NFATc1 showed altered expression levels in peripheral blood CD4+ T cells from 300 children and adults from the Asthma BRIDGE cohort (P < .05; Fig 1, F, and Table III). This cohort was described previously by Raby et al, and some of the microarray data regarding genes other than NFATc1 and IRF4 were described before. Both the PreDicta study and the Asthma BRIDGE study used the same phenotype definitions. The shorter isoform A (variant 1) lacks the C-terminal extension of approximately 245 amino acids that is present in all other NFAT proteins. This isoform of NFATc1 was found to be significantly overexpressed in peripheral blood CD4+ T cells from asthmatic patients with a positive skin test result compared with both asthmatic patients with a negative skin test result and nonasthmatic subjects with a negative skin test result (Table III). Moreover, the induced isoform D (variant 4) of NFATc1 was also found to be differentially expressed between asthmatic patients with a positive skin test result and nonasthmatic subjects with a negative skin test result (Table III). The log fold change (FC) for NFATc1 expression was 0.21 (1.2-fold increase), with an average expression of 7.44 (P = .013). The log FC for NFATc1 expression was 0.1 (1.1-fold increase), with an average expression of 7.83 (P = .013). Adding the 84 white asthmatic patients with positive skin test results to the model and adjusting for age, race, and sex (146 black plus 84 white vs 39 black subjects; female/male ratio, 127:106) did not change the NFATc1 result (log FC = 0.1, P = .018). Induction of the short isoform A of NFATc1 takes place after activation of T cells and is controlled by promoter 1 (Fig 1, A), a strong inducible promoter. This effect is autoregulated by the NFAT transcription factors. NFATc1/A is thought to be needed for exerting effector functions in activated T cells. In contrast to NFATc1/C, NFATc2, and NFATc3 proteins, the short isoform A of NFATc1 is not able to promote cell apoptosis. We presume that this function of NFATc1 leads to a prolonged survival of effector T cells in asthmatic patients with a positive skin test result. Moreover, it has been observed that NFATc1/αA (Fig 1, A) is the most prominent NFATc1 protein on receptor stimulation of peripheral B and T cells. In these cells the first activation, through the respective receptor, of the full induction of NFATc1/αA requires 24 hours. By contrast, this isoform is completely induced within a few hours after secondary stimulation. This is in line with our findings that NFATc1/A is upregulated in CD4+ T cells of asthmatic patients with a positive skin test result because these patients were already sensitized to an allergen. Moreover, we observed that NFATc1 isoform D (variant 4) was upregulated in asthmatic patients with a positive skin test result. To our knowledge, the function of NFATc1/D has not been described yet.

Table III

Comparison between different groups of children and adults with or without asthma and atopy (Asthma BRIDGE)

Group comparisons	Asthma +, skin test + (n = 251)
Asthma −, skin test + (n = 44)	P = .1
Asthma +, skin test − (n = 47)	P = .006 (NFATc1 isoform A)
Asthma −, skin test − (n = 75)	P = .002 (NFATc1 isoform A);P = .05 (NFATc1 isoform D)

For analysis using a microarray, there were 47,009 tag probes to target individual exons, including 4 exon sequences linked to specific NFATc1 isoforms. The particular isoforms of NFATc1 mRNA were as follows: NFATc1 isoform A = variant 1; NM 172390.2; NP 765978.1; NFATc1 isoform D = variant 4; NM 172388.2; NP 765976.1; Gene ID: 4772. P values for association of probes with phenotypes were adjusted by using the Benjamini-Hochberg method for multiple comparisons between groups.

In addition to NFATc1, the transcription factor interferon regulatory factor 4 (IRF4), which is encoded by the IRF4 gene, has been shown to play a role in the differentiation of various T-cell subsets known to have an effect on asthma pathology. Therefore we investigated IRF4 mRNA expression in whole blood from healthy and asthmatic children from PreDicta (Fig 2, A). We found that the asthmatic children had a significantly increased expression of IRF4 compared with that seen in healthy control subjects (Fig 2, A), especially when they also had a positive skin test result (Fig 2, B). This observation was further confirmed by analyzing total blood cells in Asthma BRIDGE asthmatic patients with positive skin test results (P = .02; Fig 2, C). IRF4 is known to be an important factor for innate and adaptive immune responses, cooperating with various other transcription factors, including NFAT family members, to act as both a transcriptional repressor and activator. Therefore it is likely that IRF4 interacts with NFATc1 to prolong the survival of effector T cells in patients with allergic asthma. Namely, IRF4 would directly promote the production of IL-4, one of the TH2-associated cytokines that contributes to asthma pathogenesis, by binding to the IL-4 promoter in cooperation with NFATc1. In support of this notion, we found reduced IFNG mRNA expression in the group of children with positive skin test results who had higher levels of NFATc1 and IRF4. Consistently, we previously reported that asthmatic mice deficient in NFATc1 in T cells (NFATc1fl/flxCD4Cre) have increased numbers of IFN-γ+CD4+ T cells in their lungs and that these cells expressed less Batf, a transcription factor essential for immunoglobulin class-switching that cooperates with IRF4 at the promoter of different genes relevant for asthma and that NFATc1fl/flxCD4Cre mice have reduced serum levels of ovalbumin (OVA)–specific IgE. Moreover, these data are also supported by our findings in the basic leucine zipper transcription factor, ATF-like (BATF)–deficient mice, which do not induce IgE, and we demonstrated T cells that produce increased IFN-γ levels in a model of allergic asthma.

Fig 2

Increased mRNA expression and IL-9 production in asthmatic patients with a positive skin test result. A,IRF4 mRNA expression analyzed by using quantitative real-time PCR in RNA isolated from whole blood (n = 13-14 children per group). B and C, Further subdivision of children shown in Fig 2, A, into healthy subjects with negative skin test results (n = 6, PreDicta, Fig 2, B; n = 104, Asthma BRIDGE, Fig 2, C) and asthmatic patients with positive skin test results (n = 8, PreDicta, Fig 2, B; n = 144, Asthma BRIDGE, Fig 2, C). D and E, PBMCs from the PreDicta children were cultured with PHA for 48 hours, and afterward, ELISA was performed for IL-9 production on cell supernatants (Fig 2, D: n = 19 healthy children and n = 20 asthmatic children; Fig 2, E: n = 8 healthy children with negative skin test results and n = 15 asthmatic children with positive skin test results). The Student t test was used to evaluate statistical significance. *P ≤ .05 and **P ≤ .01. Data are presented as means ± SEs.

Because IRF4 is also known to be crucial for IL-9 production, we also investigated IL-9 in cultured PBMCs of preschool children stimulated with PHA. We could not find any differences in IL-9 production when we compared asthmatic and healthy control children (Fig 2, D), but we observed a significant increase in IL-9 levels in the supernatants of PBMCs isolated from asthmatic children with an additional positive skin test result compared with those isolated from healthy children with a negative skin test result (Fig 2, E).

Both NFATc1 and IRF4 also positively influence IL-9 production of TH9 cells. Consistent with the increased expression of both IRF4 and NFATc1 seen in asthmatic patients with positive skin test results, we also found increased IL-9 levels in their PBMCs.

Overall, we found that in PBMCs and CD4+ T cells of asthmatic patients with a positive skin test result, expression of NFATc1 isoforms and IRF4 was increased, suggesting a fundamental role for both transcription factors in the immunologic switch in allergic asthma. In this survey we analyzed subjects from 2 different studies. Although the PreDicta study includes only 42 children, we obtained important indications regarding NFATc1, IRF4, and IL-9 expression in patients with allergic asthma. These results could be verified and confirmed in the bigger cohorts of the Asthma BRIDGE study, in which 300 asthmatic patients and 122 control subjects were included. The functional relevance of NFATc1 in asthmatic patients was supported by our observations in targeted conditional deletion of NFATc1 in T lymphocytes, where Nfatc1/A mRNA expression perfectly correlated with OVA-specific IgE levels (R = 0.94) in patients with experimental asthma (see Fig E1, D, and the Methods, Results, and Discussion sections in this article's Online Repository at www.jacionline.org) and resulted in downregulation of IL-9 (see Fig E1, B) and mast cell function (see Figs E2 and E3 and the Methods, Results, and Discussion sections in this article's Online Repository at www.jacionline.org). Therefore targeting NFATc1 in T lymphocytes might ameliorate the allergic phenotype seen in asthmatic patients.

Fig E1

Decreased IL-9 production in NFATc1fl/flxCD4Cre mice after allergen sensitization and challenge. A, Experimental design of the OVA-induced asthma model. B, IL-9 protein concentrations were measured in supernatants of purified lung CD4+ T cells from allergen-treated mice and cultured for 24 hours with α-CD3 and α-CD28 antibodies (n = 7-11 mice per group). C,Irf4 mRNA expression measured by using quantitative real-time PCR in isolated lung CD4+ T cells (n = 8-13 mice per group). D, Correlation between Nfatc1/A mRNA expression in lung CD4+ T cells and OVA-specific IgE levels in serum of Nfatc1fl/fl OVA-treated mice (n = 6). E, Correlation between Irf4 mRNA expression in lung CD4+ T cells and OVA-specific IgE in serum of Nfatc1fl/fl OVA mice (n = 6). The Student t test was used to evaluate statistical significance. *P ≤ .05. Data are presented as means ± SEMs.

Fig E2

Decreased mast cell numbers and activation in NFATc1fl/flxCD4Cre mice. A, Mast cell numbers (c-kit+FcεRI+CD123+ cells) were analyzed by using flow cytometry in the lungs of NFATc1fl/fl control mice and NFATc1fl/flx CD4Cre mice (n = 4-11 mice per group). B, IgE serum levels were measured with ELISA (n = 12-19 mice per group). C, Experimental design for bone marrow–derived mast cell (BMMC) differentiation (upper panel) and histamine release (lower panel). Histamine release was measured by means of ELISA (lower panel; n = 4 mice per group). Mast cells differentiated from Nfatc1fl/flxCD4Cre mice received serum from OVA-sensitized and challenged Nfatc1fl/flxCD4Cre mice, whereas mast cells from Nfatc1fl/fl mice received serum from OVA-sensitized and challenged Nfatc1fl/fl mice. Statistical significances in this figure were evaluated with the Student t test. *P ≤ .05, **P ≤ .01, and ***P ≤ .001. Data are presented as means ± SEMs.

Fig E3

Mast cell differentiation. A, Flow cytometric analysis of bone marrow–derived mast cell differentiation, as described in the Methods section in this article's Online Repository. FACS analysis for FcεRI+c-kit+ cells was performed every week to monitor the differentiation status of the cultured cells. B, Corresponding dot plots from the first and fourth weeks of mast cell differentiation are shown.