Visual Abstract

RNA-binding protein HuR (ELAVL1) is a master regulator of gene expression in human pathophysiology. Its dysregulation plays an important role in many diseases. We hypothesized that HuR plays an important role in Th2 inflammation in asthma in both mouse and human. To address this, we used a model of airway inflammation in a T cell–specific knockout mouse model, distal lck-Cre HuRfl/fl, as well as small molecule inhibitors in human peripheral blood-derived CD4+ T cells. Peripheral CD4+ T cells were isolated from 26 healthy control subjects and 45 asthmatics (36 type 2 high and 9 non–type 2 high, determined by blood eosinophil levels and fraction of exhaled NO). Our mouse data showed conditional ablation of HuR in T cell–abrogated Th2 differentiation, cytokine production, and lung inflammation. Studies using human T cells showed that HuR protein levels in CD4+ T cells were significantly higher in asthmatics compared with healthy control subjects. The expression and secretion of Th2 cytokines were significantly higher in asthmatics compared with control subjects. AMP-activated protein kinase activator treatment reduced the expression of several cytokines in both type 2 high and non–type 2 high asthma groups. However, the effects of CMLD-2 (a HuR-specific inhibitor) were more specific to endotype-defining cytokines in type 2 high asthmatics. Taken together, these data suggest that HuR plays a permissive role in both allergen and non–allergen-driven airway inflammation by regulating key genes, and that interfering with its function may be a novel method of asthma treatment.

Asthma is a chronic inflammatory airway disease that exists in multiple subendotypes. It is estimated that >300 million people have asthma worldwide, with a majority of patients having the type 2 high endotype. The type 2 high endotype is associated with increases in Th2 cytokine activity, including IL-4, IL-5, and IL-13 (13). These cytokines act in concert to promote airway eosinophilia, IgE synthesis, mucous overproduction, and bronchial hyperresponsiveness (4, 5). The presence of Th2 cytokines in the airway is an established mechanism in (type 2) asthma, and the use of inhaled corticosteroids is the cornerstone of treatment to decrease cytokine levels in the lung (5, 6). Previously, Wenzel et al. (7) divided corticosteroid-dependent severe asthma into two different subtypes based on the presence of airway eosinophilia, which subsequently led to categorizing type 2 asthma into two endotypes, eosinophilic and noneosinophilic. This classification is relevant to differences in treatment responsiveness to existing or newly developed biological medications (8, 9). Clinical trials targeting a variety of biological targets (IgE, IL-5, and IL-4/IL-13 or their receptors) have resulted in the approval of several of these novel biologics for treating asthma (9, 10). These biologic agents have revolutionized the approach to asthma therapeutics. With the development of these novel biological drugs, asthma symptoms are controlled in some patients who do not respond to conventional asthma therapy (9). However, a significant proportion of patients remain who do not respond completely to these biologically targeted therapies (11, 12). Furthermore, the use of these biologics is restricted to patients whose symptoms are not controlled by previously established therapeutics (13). Yet, as with many asthma therapies, including biologics, when they are stopped, the symptoms return. Studies are currently in progress to develop discrete biomarkers and genetic profiling to further individualize treatments in asthmatic patients. Despite these extensive advances, severe asthma still remains a difficult-to-treat chronic respiratory disease, suggesting diverse molecular mechanisms in different asthmatic populations. Understanding the mechanisms underlying the production of the proinflammatory cytokines may, therefore, aid in the identification of more effective asthma therapeutics.

Posttranscriptional gene regulation by RNA-binding proteins (RBPs) is an important mechanism controlling proinflammatory gene expression. One of the best characterized RBPs is HuR (encoded by ELAVL1), which is a master regulator of proinflammatory genes involved in the pathophysiology of numerous diseases (1416). HuR can influence stability and/or translation of mRNA via interacting with their 3′ untranslated region (UTR) adenylate and uridylate-rich elements (17). By stabilizing target mRNAs, HuR plays an important role in biological activities during cell-cycle development, immune function and inflammation, and apoptosis; it is also involved in oncogenic activities (18). HuR functions in the nucleus by regulating the splicing of RNA and taking part in nuclear export, or in the cytosol by binding to uridylate-rich elements in the 3′ UTR of target mRNAs and therefore protecting them against degradation (18).

We previously reported posttranscriptional regulation of proinflammatory gene expression by HuR in CD4+ T cells in animal (16, 17, 1921) and human studies (21, 22). Employing different conditional HuR knockout (KO) mouse models, we demonstrated the regulatory effect of HuR on activation and differentiation of CD4+ Th subsets (19, 23), maintaining IL-2 homeostasis in CD4+ T cells (17) and cytokine production from Th2-polarized cells (20). In this study, we first demonstrated the role of HuR in murine lung inflammation using an OVA-induced mouse model.

In our previous studies, Th2-skewed cells from peripheral blood from healthy control subjects (22) and the Jurkat T cell line (21, 22) also showed the regulatory effect of HuR in gene expression of Th2 cytokines and/or GATA-3 (21), which is an essential transcriptional factor for Th2 polarization and differentiation (21, 24, 25). However, the role of HuR in proinflammatory responses in CD4+ T cells from asthmatic populations is not well understood. We set out to investigate these mechanisms because human genes are not necessarily regulated by the same mechanisms as their murine counterparts. In this article, for the first time to our knowledge, we show the role of HuR in regulating cytokine production and GATA3 in activated peripheral CD4+ T cells from type 2 high and non–type 2 high asthmatics. We also demonstrate that interfering with HuR action using two different reagents (an AMPK activator, 5-aminoimidazole-4-carboxamide riboside [AICAR], also known as acadesine, and a HuR-specific small molecule inhibitor [CMLD-2]) can ameliorate these proinflammatory responses. Our data strongly support HuR as a novel therapeutic target to treat asthmatic lung inflammation.

Generation of distal lck-Cre ROSA HuRfl/fl

The generation of HuR floxed mice (HuRfl/fl) in our laboratory was described previously (17, 20). HuRfl/fl mice were crossed to distal lck-Cre and ROSA-YFP mice to generate distal lck-Cre ROSA HuRfl/fl mice (HuR KO). In these mice, HuR-ablated T cells are labeled with a YFP marker. All mice used were on a C57BL/6 background. All animal procedures were performed under the U.S. National Institutes of Health guidelines and were approved by the University of Missouri and University of Michigan Committees on the Use and Care of Animals.

Murine allergic airway inflammatory model and experimental studies

Allergic airway inflammation in HuR KO and ROSA HuRfl/fl littermate control mice was induced using the standard OVA-alum–induced allergic inflammation protocol as previously described (20). Fig. 1A summarizes the sensitization and challenge protocol for the OVA-inducing asthma model in this study. Six- to eight-week-old female HuR KO or ROSA HuRfl/fl (littermate control) mice were immunized i.p. with 100 µg of chicken OVA grade V (Sigma-Aldrich) and 1 mg aluminum hydroxide (Thermo Scientific) on days 0 and 7. Mice were intranasally challenged with 1% OVA in PBS in a nebulization chamber for 30 min daily on days 12–15 for four doses. Sham controls received intranasal challenge using only PBS. Twenty-four hours after the last challenge, mice were sacrificed. Mouse sera were collected by retro-orbital bleeding using heparinized sterile pipettes. Mouse lungs were lavaged with 1 ml of sterile PBS. Bronchoalveolar lavage (BAL) fluid (BALF) was harvested for cell counting using a hemocytometer. BALF was then centrifuged at 4000 rpm using a tabletop centrifuge for 5 min at 4°C. Supernatants were removed and saved for murine IL-4 and IL-13 cytokine measurement by ELISA. Cell pellets from BALF were resuspended in 200 µl of 2% BSA in PBS, and cytospin was performed on slides. Diff-Quick staining was performed to differentiate cells. Accessory lobes of the lungs were isolated and digested with collagenase A and DNase I to analyze the presence of immune cells in the lungs. Mouse lungs were inflated and fixed with buffered zinc formalin. Lungs were sectioned and stained with H&E using a standard procedure. The pattern of lung inflammation in mice was evaluated by H&E staining in tissue, as well as cell counting and ELISA in BALFs.

Study population

A total of 45 asthmatics (with the confirmed diagnosis of asthma by physician) and 26 healthy control subjects were enrolled in the study. Subjects were randomly assigned to different groups for experiments, as per Institutional Review Board protocol. Due to limitations of blood draw volumes, it was not feasible to perform all the experiments with all subjects. Demographic characteristics of the study population are presented in Table I. Clinical data, including lung function tests, fraction of exhaled NO (FeNO), peripheral blood eosinophil counts, and total IgE from the asthmatic patients are shown in Table II. Type 2 high asthma (n = 36) was defined for the purposes of this study as having either blood eosinophil counts ≥ 300 cells/μl or having FeNO levels ≥ 25 parts per billion, also taking into account high serum IgE levels (>30 IU, which is associated with type 2 inflammation; therefore, this cutoff value was also used for determining the type 2 high asthma phenotype). Those asthmatics without these criteria were classified as non–type 2 high asthmatics (n = 9). Subjects undergoing treatment with biologics were excluded from this study. The study protocol was approved by the Institutional Review Board of the University of Michigan. The written informed consent was obtained from all participants before any study-related activities.

Peripheral CD4+ T cell isolation

Peripheral whole blood samples (up to 150 ml) were acquired from study subjects in heparinized tubes. PBMCs were isolated by Ficoll-Hypaque (Sigma) density gradient centrifugation. In brief, 16 ml of diluted blood (1:1 in PBS) was slowly added over 12 ml Ficoll-Hypaque solution and centrifuged at 400 × g for 40 min at 20°C (low acceleration, no brake). Intermediate, white flocculent cells (mononuclear cells) were collected after plasma layer isolation. The CD4+ T cells were then isolated from these mononuclear cells suspension by positive selection using the Miltenyi MACS magnetic bead isolation system according to the manufacturer’s protocol (Miltenyi Biotec, Bergisch Gladbach, Germany). The purity of the isolated CD4+ T cells was >95%, as determined by flow cytometry.

Western blot analysis

Isolated CD4+ T cells, from some randomly selected patients, were lysed in Pierce RIPA Lysis Buffer (Thermo Scientific) supplemented with 1× complete protease inhibitor mixture (Roche) after washing with cold PBS. After denaturing the cell lysates, 10 μg of proteins was loaded per lane on 4–20% SDS-PAGE gels (Bio-Rad) and transferred onto nitrocellulose membranes (Bio-Rad). The membranes were blocked with Odyssey blocking buffer for 1 h at room temperature, followed by incubation at 4°C overnight with primary Abs, including anti-HuR clone 3A2 (1 μg/ml), Cyclophilin B (D1V5J) Rabbit mAb (Cell Signaling). Membranes were washed with TBS-T and then incubated with fluorescently labeled IRDye secondary Abs (LI-COR), including IRDye 680RD Goat anti-Rabbit IgG and IRDye 800CW Donkey anti-Mouse IgG for 1 h at room temperature. All primary and secondary Abs were diluted in Odyssey blocking buffer supplemented with 0.2% Tween 20. After TBS-T washes, digital fluorescence of membranes was visualized by detecting signals at 700- and/or 800-nm channels using the OdysseyCLx Imaging System (LI-COR). Quantitation of protein was done using Image Studio Lite v5.2 (LI-COR) and expressed as relative arbitrary units.

CD4+ T cell activation and inhibition interventions

Enriched CD4+ T cells were cultured in RPMI 1640 Complete Medium supplemented with 10% FBS, 2 mM l-glutamine, 1 mM sodium pyruvate (all from Life Technologies), 50 µg/ml gentamicin sulfate (IBI Scientific), and 0.05 mM 2-ME (Fisher). CD4+ T cells were activated for 4 d using immobilized anti-CD3 (5 µg/ml) and anti-CD28 (2 µg/ml) (both from Invitrogen) either by precoating the culture plate or by addition of anti-CD3/CD28 Dynabeads (Invitrogen) as described previously (17, 19). For studying the inhibitory effect of AICAR (Sigma-Aldrich) or CMLD-2 (Millipore), CD4+ T cells were treated with AICAR (1 mM) or CMLD-2 (10 μM) or their vehicle control (3% H2O or 0.35% DMSO, respectively) starting 2 h before activation with anti-CD3/CD28. On day 4, activated cells were harvested for protein and mRNA studies, and the culture supernatant was collected for measuring cytokine release. For intracellular staining studies, activated CD4+ T cells were restimulated with PMA (50 ng/ml) and Ionomycin (1 μg/ml) (both reagents from Sigma) on day 4 for 4 h in the presence of brefeldin A (3 μg/ml) (from BD Biosciences).

Intracellular staining and flow cytometry

Activated CD4+ T cells were harvested after restimulation on day 4 (as described earlier) and washed with PBS. The Fc receptors were then blocked using Fc Receptor Blocking Solution (Human TruStain FcX; BioLegend) in 100 μl of FACS buffer for 15 min on ice. The cells were then treated with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies) in PBS for 30 min according to the manufacturer’s instructions. After washing with PBS, the cells were stained with CD4+ marker Ab in FACS buffer for 30 min at 4°C. Then the cells were washed with PBS and permeabilized with BioLegend’s fix/permeabilizing buffer kit, according to the manufacturer’s guideline. Cells were subsequently stained for intracellular markers, including cytokines (IL-4, IL-5, IL-13, IL-17, IFNγ) and GATA3 and HuR (detected by a PE-labeled secondary Ab) in fix/perm wash buffer for 30 min at 4°C. After final wash, the stained cells were analyzed for expression of the targeted markers using an Attune NxT Flow Cytometer (Invitrogen). ArC Amine Reactive Compensation Bead Kit (Invitrogen) was used for compensating LIVE/DEAD Fixable dead cell Aqua viability stain, and UltraComp eBeads Plus Compensation Beads (Invitrogen) were used for compensating all the Fluorochrome-conjugated Abs. Data were analyzed using FlowJo software (Tree Star, Ashland, OR). More than 5 × 104 live cells were examined by forward and side light scatter properties. Fluorescence minus one controls were used for gating positive subpopulations.

LEGENDplex bead-based multiplex assay

LEGENDplex HU Th Cytokine Panel (13-plex) from BioLegend was used to quantify cytokine levels in the culture supernatant, using fluorescence-encoded beads on flow cytometer according to the manufacturer’s instructions. This panel allowed simultaneous quantification of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22, IFNγ, and TNF-α.

RNA isolation and real-time quantitative PCR

RNA isolation was performed using TRIzol extraction, and cDNA was made by SuperScript III Reverse Transcriptase according to the manufacturer’s protocol (Invitrogen). Quantitative RT-PCR was performed using Platinum SYBR Green Universal (Invitrogen) as we described previously (26) on QuantStudio 3 Real-Time PCR Systems (Applied Biosystems, Foster City, CA). Data were analyzed by ΔΔCT to represent data as fold change relative to the β-actin housekeeping gene or the mRNA ratio to the reference gene. The mRNA primers (Integrated DNA Technologies) used for amplification were as follows:

HuR, 5′-ATGAAGACCACATGGCCGAAGACT-3′ (forward), 5′-AGTTCACAAAGCCATAGCCCAAGC-3′ (reverse); β-Actin, 5′-TCAGAAGGATTCCTATGTGGGCGA-3′ (forward), 5′-TTTCTCCATGTCGTCCCAGTTGGT-3′ (reverse); GATA3, 5′-TGTCTGCAGCCAGGAGAGC-3′ (forward), 5′-ATGCATCAAACAACTGTGGCCA-3′ (reverse); IL-4, 5′-CAGACATCTTTGCTGCCTCC-3′ (forward), 5′-GTGTCCTTCTCATGGTGGCT-3′ (reverse); IL-5, 5′-AGCTGCCTACGTGTATGCCA-3′ (forward), 5′-GCAGTGCCAAGGTCTCTTTCA-3′ (reverse); IL-13, 5′-TGAGGAGCTGGTCAACATCA-3′ (forward), 5′-CAGGTTGATGCTCCATACCAT-3′ (reverse); IFNγ, 5′-ACAGTTCAGCCATCACTTGGA-3′ (forward), 5′-CTAATTATTCGGTAACTGACTTGA-3′ (reverse); and RORγt, 5′-GCTGAGAAGGACAGGGAGC-3′ (forward), 5′-GACGACTTGTCCCCACAGATT-3′ (reverse).

mRNA stability measurement by actinomycin D

On day 4 postactivation, CD4+ T cells were treated with 3 μg/ml actinomycin D (Act D) to stop nascent mRNA transcription. Cells were collected at 0, 1, 2, 3, and 4 h after Act D treatment, the remaining RNA was isolated from the cells using TRIzol extraction, and real-time quantitative PCR was performed as we described previously (17, 21, 22, 27). The amount of RNA at 0 h was set to 100%, and the percentage of remaining RNA at 0–4 h was plotted using a semilog scale. A half-life for mRNA decay was calculated using the best fit values in GraphPad Prism software.

For statistical analysis, GraphPad Prism version 8 software (GraphPad Software, La Jolla, CA) was used. All values are expressed as means ± SD. Data were analyzed by two-tailed Student t test, paired t test, ANOVA with Tukey, or Sidak multiple comparison test, as appropriate. Differences were considered significant when p < 0.05.

We first asked whether there are physiological consequences of T cell–specific ablation of HuR using an OVA model of airway inflammation. The immunization and challenge protocol of the mice were described earlier and shown in (Fig. 1A. As shown in (Fig. 1, immunized HuR-deficient mice developed significantly less severe allergic inflammation as compared with immunized ROSA HuRfl/fl controls. Major reductions in the total cell numbers and eosinophilia in the lungs of immunized HuR KO mice were observed (Fig. 1B, 1C, black bars). IL-13 cytokine levels in BALF from immunized HuR KO mice were significantly lower than those of immunized controls (Fig. 1D). We also observed a trend toward reduced IL-4 in BALF of HuR KO mice; however, this was not statistically significant (Fig. 1D). In addition, immunized HuR KO mice had slightly decreased, although not statistically significant, total serum IgE levels (Fig. 1E). Remarkably, immunized HuR KO mice have comparable levels of lung inflammation as sham-challenged mice (Fig. 1B, black versus gray bars, and (Fig. 1F, H&E staining), indicating alleviation of lung inflammation.

FIGURE 1.

HuR ablation in CD4+ T cells alleviates allergic airway inflammation. (A) Sensitization and challenge protocol for murine model of OVA-induced asthma model. (B) Total cell numbers recovered from BALF of immunized HuR KO mice, immunized control mice (ROSA HuRfl/fl), and sham-challenged mice (HuR KO and ROSA HuRfl/fl). (C) Immune cell composition of cells recovered from BALF of immunized HuR KO mice, immunized control mice, sham-challenged HuR KO mice, and sham-challenged control mice. (D) IL-4 and IL-13 levels in BALF of immunized HuR KO mice, immunized control mice, and sham-challenged control mice as detected by ELISA. (E) Serum total IgE levels of immunized HuR KO mice, immunized control mice, and sham-challenged control mice as measured by ELISA. (F) Representative H&E staining (original magnification ×100) of lung sections from mice described earlier. Representative of at least five mice per group in four individual experiments. BALF IL-4, IL-13 (D), and IgE (E) levels from sham-challenged HuR KO mice were below the level of detection and, as such, were not included. The values are presented as mean ± SD and were analyzed by one-way ANOVA with the Tukey test for multiple comparisons between the groups. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 1.

HuR ablation in CD4+ T cells alleviates allergic airway inflammation. (A) Sensitization and challenge protocol for murine model of OVA-induced asthma model. (B) Total cell numbers recovered from BALF of immunized HuR KO mice, immunized control mice (ROSA HuRfl/fl), and sham-challenged mice (HuR KO and ROSA HuRfl/fl). (C) Immune cell composition of cells recovered from BALF of immunized HuR KO mice, immunized control mice, sham-challenged HuR KO mice, and sham-challenged control mice. (D) IL-4 and IL-13 levels in BALF of immunized HuR KO mice, immunized control mice, and sham-challenged control mice as detected by ELISA. (E) Serum total IgE levels of immunized HuR KO mice, immunized control mice, and sham-challenged control mice as measured by ELISA. (F) Representative H&E staining (original magnification ×100) of lung sections from mice described earlier. Representative of at least five mice per group in four individual experiments. BALF IL-4, IL-13 (D), and IgE (E) levels from sham-challenged HuR KO mice were below the level of detection and, as such, were not included. The values are presented as mean ± SD and were analyzed by one-way ANOVA with the Tukey test for multiple comparisons between the groups. *p < 0.05, **p < 0.01, ***p < 0.001.

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Table I.

Study population characteristics

Sex, n (%)Age, y, Mean ± SD (Minimum to Maximum)Race, n (%)
Asthmatics (45 cases) Male: 13 (28.9) 47.25 ± 13.83 (18–69) White: 33 (73.3) 
 Female: 32 (71.1)  African American: 11 (24.4) 
   Others: 1 (2.2) 
Control subjects (26 cases) Male: 6 (23.1) 40.50 ± 11.59 (18–59) White: 19 (73.1) 
 Female: 20 (76.9)  African American: 2 (7.7) 
   Others: 5 (19.2) 
Sex, n (%)Age, y, Mean ± SD (Minimum to Maximum)Race, n (%)
Asthmatics (45 cases) Male: 13 (28.9) 47.25 ± 13.83 (18–69) White: 33 (73.3) 
 Female: 32 (71.1)  African American: 11 (24.4) 
   Others: 1 (2.2) 
Control subjects (26 cases) Male: 6 (23.1) 40.50 ± 11.59 (18–59) White: 19 (73.1) 
 Female: 20 (76.9)  African American: 2 (7.7) 
   Others: 5 (19.2) 
Table II.

Asthmatics lung function and paraclinical data

FEV1% Predicted, Median (Min–Max)FEV1/FVC %, Median (Min–Max)FeNO ppb, Median (Min–Max)Blood EOS Cells/µl, Median (Min–Max)Total IgE, kU/L, Median (Min–Max)
Non–type 2 high (9 cases) 86 (59–98) 81 (56–86) 15 (<5–20) 200 (0–200) 27 (6–71) 
Type 2 high (36 cases) 79 (27–121) 74 (49–95) 38 (6–245) 300 (100–2800) 155 (6–1914) 
FEV1% Predicted, Median (Min–Max)FEV1/FVC %, Median (Min–Max)FeNO ppb, Median (Min–Max)Blood EOS Cells/µl, Median (Min–Max)Total IgE, kU/L, Median (Min–Max)
Non–type 2 high (9 cases) 86 (59–98) 81 (56–86) 15 (<5–20) 200 (0–200) 27 (6–71) 
Type 2 high (36 cases) 79 (27–121) 74 (49–95) 38 (6–245) 300 (100–2800) 155 (6–1914) 

EOS, eosinophils; FeNO, fraction of exhaled NO; FEV1, forced expiratory volume in 1 s; FVC, forced vital capacity; Min–Max, minimum to maximum; NO; ppb, parts per billion.

We then asked whether the lack of airway inflammation was due to defective CD4+ T cell trafficking. We isolated CD4+ T cells from the lungs and mediastinal lymph nodes of challenged mice and ascertained their phenotype. We found that the majority (64%) of cells that traffic to the lungs are CD4+ HuRlo and express greater levels of IL-2, as compared with HuRfl/fl and WT mice (data not shown). In summary, these data indicate HuR KO mice have impaired OVA-induced airway inflammation.

To study the expression levels of HuR in peripheral CD4+ T cells in asthmatic subjects (type 2 high and non–type 2 high) versus healthy control subjects, we isolated peripheral blood CD4+ T cells from these three groups (Tables I, II). HuR expression in nonactivated CD4+ T cells was determined in randomly selected subjects by Western blot to quantitate baseline HuR levels. As shown in (Fig. 2, there was significantly higher expression of HuR protein in nonactivated peripheral CD4+ T cells from type 2 high asthmatics compared with both non–type 2 high asthmatics and control subjects.

FIGURE 2.

Higher expression of HuR protein in peripheral CD4+ T cells in type 2 high asthmatics compared with non–type 2 high asthmatics and healthy control subjects. (A and B) Isolated peripheral blood CD4+ T cells from 7 healthy control individuals and 10 asthmatics (5 non–type 2 high and 5 type 2 high) were lysed in Pierce RIPA Lysis buffer. HuR expression from 10 µg of the lysate was evaluated by Western blot. (A) The ratio of HuR protein (36 kDa) to cyclophilin B (20 kDa), as the internal control, from 7 healthy control subjects and 10 asthmatics (5 non–type 2 high and 5 type 2 high). The values are presented as mean ± SD and were analyzed by one-way ANOVA with the Tukey test for multiple comparisons between the groups. **p < 0.01, ***p < 0.001. (B) Representative blot including four healthy control subjects, three type 2 high asthmatics, and three non–type 2 high asthmatics, as also indicated by the intensity ratio for each sample on the image. a.u., arbitrary units.

FIGURE 2.

Higher expression of HuR protein in peripheral CD4+ T cells in type 2 high asthmatics compared with non–type 2 high asthmatics and healthy control subjects. (A and B) Isolated peripheral blood CD4+ T cells from 7 healthy control individuals and 10 asthmatics (5 non–type 2 high and 5 type 2 high) were lysed in Pierce RIPA Lysis buffer. HuR expression from 10 µg of the lysate was evaluated by Western blot. (A) The ratio of HuR protein (36 kDa) to cyclophilin B (20 kDa), as the internal control, from 7 healthy control subjects and 10 asthmatics (5 non–type 2 high and 5 type 2 high). The values are presented as mean ± SD and were analyzed by one-way ANOVA with the Tukey test for multiple comparisons between the groups. **p < 0.01, ***p < 0.001. (B) Representative blot including four healthy control subjects, three type 2 high asthmatics, and three non–type 2 high asthmatics, as also indicated by the intensity ratio for each sample on the image. a.u., arbitrary units.

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We have previously shown that HuR plays an important role in T cell activation (17, 19, 20), as well as in the regulation of Th2 cytokines and GATA3 gene expression (20, 21). Specifically, HuR controls the mRNA half-lives of GATA3 and Th2 cytokines (21). Therefore, in this study, we investigated the expression levels of Th2 cytokines and GATA3 after activation of peripheral CD4+ T cells. The isolated CD4+ T cells from peripheral blood of healthy control subjects, type 2 high asthmatics, and non–type 2 high asthmatics were activated, and cytokine and GATA3 expression were quantitated using flow cytometry.

As shown in (Fig. 3, Th2 cytokines (IL-4, IL-5, and IL-13) (Fig. 3A–C) and GATA3 (Fig. 3F) were expressed at significantly higher levels in activated CD4+ T cells from type 2 high asthmatics compared with those from the control subjects. However, there was no significant difference observed between the levels of IFNγ (Fig. 3D) and IL-17 (Fig. 3E) in activated CD4+ T cells between these two groups. The expression of two Th2 cytokines, including IL-5 and IL-13, also showed significant increases in CD4+ T cells from non–type 2 asthmatics compared with those in the healthy control group (Fig. 3B, 3C, respectively). The expression of IL-17 was also significantly higher in non–type 2 high asthmatics compared with that in healthy control subjects (Fig. 3E). There were no consistent differences between type 2 high and non–type 2 high asthmatics. Together, these data show that type 2 high asthmatics express greater levels of GATA3 and Th2 cytokines, whereas non–type 2 high asthmatics have higher levels of IL-5, IL-13, and IL-17 (perhaps a signature of the predominant Th2/Th17 asthma subtypes) compared with healthy control subjects. IFNγ levels are similar among all three groups (Fig. 3D).

FIGURE 3.

Higher expression of Th2 cytokines and GATA3 (AF) and increased cytokine secretion (GK) in activated peripheral CD4+ T cells in type 2 high and non–type 2 high asthmatics compared with healthy control subjects. (A–F) Isolated peripheral blood CD4+ T cells from 10 healthy control subjects and 29 asthmatics (9 non–type 2 high and 20 type 2 high) were stimulated with anti-CD3/CD28 for 4 d and then treated for an additional 4 h with PMA (50 ng/ml) and Ionomycin (1 µg/ml) in the presence of brefeldin A (3 µg/ml). Expressions of proinflammatory cytokines (A–E) and GATA3 (F) were evaluated by flow cytometry and shown as mean fluorescence intensity (MFI) values. (G–K) The secretion of the cytokines from the activated CD4+ T cells in 7 healthy control subjects and 10 asthmatics (5 non–type 2 high and 5 type 2 high) was evaluated by BioLegend’s LEGENDplex bead-based immunoassay. The values are presented as mean ± SD and were analyzed by one-way ANOVA with the Tukey test for multiple comparisons between the groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 3.

Higher expression of Th2 cytokines and GATA3 (AF) and increased cytokine secretion (GK) in activated peripheral CD4+ T cells in type 2 high and non–type 2 high asthmatics compared with healthy control subjects. (A–F) Isolated peripheral blood CD4+ T cells from 10 healthy control subjects and 29 asthmatics (9 non–type 2 high and 20 type 2 high) were stimulated with anti-CD3/CD28 for 4 d and then treated for an additional 4 h with PMA (50 ng/ml) and Ionomycin (1 µg/ml) in the presence of brefeldin A (3 µg/ml). Expressions of proinflammatory cytokines (A–E) and GATA3 (F) were evaluated by flow cytometry and shown as mean fluorescence intensity (MFI) values. (G–K) The secretion of the cytokines from the activated CD4+ T cells in 7 healthy control subjects and 10 asthmatics (5 non–type 2 high and 5 type 2 high) was evaluated by BioLegend’s LEGENDplex bead-based immunoassay. The values are presented as mean ± SD and were analyzed by one-way ANOVA with the Tukey test for multiple comparisons between the groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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Secreted levels of cytokine are more physiologically relevant to disease processes compared with intracellular levels. To determine whether the secreted cytokine levels from activated CD4+ T cells are consistent with their intracellular expression levels, we used BioLegend’s LEGENDplex bead-based immunoassay. This allowed for simultaneous quantification of multiple soluble cytokines (13 targets) in supernatant from activated CD4+ T cells. Supplemental Fig. 1 shows the secretion levels of 13 cytokines by peripheral CD4+ T cells from healthy control subjects and compares the levels of these cytokines before and after stimulation (activation) with anti-CD3/CD28 for 4 d. The secretion of nearly all cytokines (except IL-6) significantly increased after stimulation (Supplemental Fig. 1). We then compared the secretion of cytokines by activated peripheral CD4+ T cells from type 2 high and non–type 2 high asthmatics and control subjects. As shown in (Fig. 3, the levels of secreted IL-5 and IL-13 (Fig. 3H, 3I, respectively) were significantly higher in supernatants from cultures of activated CD4+ T cells from type 2 high asthmatics, as compared with those from the healthy control group (Fig. 3H, 3I). The levels of secreted IL-4, IL-13, and IL-17 were significantly higher in non–type 2 high asthmatics compared with the healthy control group (Fig. 3G, 3I, 3K). Surprisingly, there was a significantly higher level of secreted IFNγ in both non–type 2 high and type 2 high asthmatics, compared with those in the healthy control group. There were no consistent differences between type 2 high and non–type 2 high asthmatics.

We hypothesized that HuR controls and regulates the expression of Th2 cytokines and GATA3 by controlling the stability and translation of their mRNAs. Therefore, we would predict that HuR inhibition should alter the production of these proteins. We initially used an AMPK (AMP-activated protein kinase) activator reagent (AICAR, also known as acadesine), which has been previously described as interfering with HuR function (28, 29). We asked whether AICAR pretreatment of CD4+ T cells during activation may diminish HuR activity and thereby alter proinflammatory responses in type 2 high and non–type 2 high asthmatics.

As shown in Supplemental Fig. 2, AICAR treatment significantly decreased the levels of several Th2 cytokines and GATA3 in cells from type 2 high and non–type 2 high asthmatics (Supplemental Fig. 2A–C, 2F). There were no observed differences in IFNγ and IL-17 expression in cells from either type 2 high or non–type 2 high asthmatics, compared with vehicle control cultures (Supplemental Fig. 2D, 2E). We conclude that blocking HuR in a nonspecific manner results in decreases in GATA3 and Th2 cytokine expression.

We further investigated the levels of cytokine secretion by activated CD4+ T cells after pretreatment with AICAR or vehicle. CD4+ T cells from either type 2 high or non–type 2 high asthmatics were pretreated with AICAR and, after activation, levels of secreted cytokines were quantitated using LEGENDplex bead-based immunoassay. Data are shown in Supplemental Fig. 2G–K and expressed as a percentage of vehicle treatment. Given their importance in Th2, Th1, and Th17 cells, respectively, we focused on IL-4, IL-5, IL-13, IFNγ, and IL-17. As shown in Supplemental Fig. 2, Th2 cytokines (except for IL-4) and IFNγ levels showed significant decreases in the AICAR-treated group compared with the vehicle control. Interestingly, IL-13 levels (Supplemental Fig. 2I) in the non–type 2 high asthma group did not show significant decreases after AICAR treatment. We conclude that AICAR treatment results in altered cytokine expression in both non–type 2 high and type 2 high asthmatics, although there are marked differences between the two endotypes in their susceptibility to suppression.

Because the function of AICAR is not specific to HuR, we evaluated whether a HuR-specific small molecule inhibitor, CMLD-2, can interfere with relevant cytokine expression in CD4+ T cells from type 2 high and non–type 2 high subjects. CD4+ T cells were pretreated with CMLD-2 and activated with anti-CD3/CD28. After restimulation, we measured intracellular cytokines and GATA3 expression levels by flow cytometry.

(Fig. 4A–F compares the effect of CMLD-2 on intracellular expression levels of cytokines and GATA3 in activated CD4+ T cells from type 2 high and non–type 2 high asthmatics. In contrast with the broader effect of the AICAR (Supplemental Fig. 2A–F), CMLD-2 significantly decreased the expression levels of IL-5, IL-13, and GATA3 (Fig. 4B, 4C, 4F, respectively) only in type 2 high asthmatics, suggesting the specific inhibitory effect of CMLD-2 compared with AICAR. Other cytokine levels did not show significant differences between CMLD-2 and the vehicle-treated group in either type 2 high or non–type 2 high asthmatics. We conclude that specific HuR inhibition results in significant decreases in IL-5, IL-13, and GATA3 expression only in type 2 high asthma. Expressions of IL-4, INF-γ, and IL-17 were not significantly affected by CMLD-2 treatment.

FIGURE 4.

Decreased expression of Th2 cytokines and GATA3 after CMLD-2 treatment (AF) and reduced levels of cytokine secretion (GK) in activated peripheral CD4+ T cells from type 2 high and non–type 2 high asthmatics. (A–F) Isolated peripheral blood CD4+ T cells from nine non–type 2 high and nine type 2 high asthmatics were incubated with CMLD-2 (10 µg/ml) or vehicle for 2 h and then stimulated with anti-CD3/CD28 for 4 d. The cells were then treated for an additional 4 h by PMA (50 ng/ml) and Ionomycin (1 µg/ml) in the presence of brefeldin A (3 µg/ml). Expressions of proinflammatory cytokines (A–E) and GATA3 (F) were evaluated by flow cytometry and shown by the percentage of positive cells. (G–K) The secretion of the cytokines from the activated CD4+ T cells in six non–type 2 high and nine type 2 high asthmatics was evaluated by BioLegend’s LEGENDplex bead-based immunoassay. The values are presented as mean ± SD and were analyzed by two-way ANOVA with the Sidak test for comparisons within each group of non–type 2 high and type 2 high asthmatics. *p < 0.05, **p < 0.01.

FIGURE 4.

Decreased expression of Th2 cytokines and GATA3 after CMLD-2 treatment (AF) and reduced levels of cytokine secretion (GK) in activated peripheral CD4+ T cells from type 2 high and non–type 2 high asthmatics. (A–F) Isolated peripheral blood CD4+ T cells from nine non–type 2 high and nine type 2 high asthmatics were incubated with CMLD-2 (10 µg/ml) or vehicle for 2 h and then stimulated with anti-CD3/CD28 for 4 d. The cells were then treated for an additional 4 h by PMA (50 ng/ml) and Ionomycin (1 µg/ml) in the presence of brefeldin A (3 µg/ml). Expressions of proinflammatory cytokines (A–E) and GATA3 (F) were evaluated by flow cytometry and shown by the percentage of positive cells. (G–K) The secretion of the cytokines from the activated CD4+ T cells in six non–type 2 high and nine type 2 high asthmatics was evaluated by BioLegend’s LEGENDplex bead-based immunoassay. The values are presented as mean ± SD and were analyzed by two-way ANOVA with the Sidak test for comparisons within each group of non–type 2 high and type 2 high asthmatics. *p < 0.05, **p < 0.01.

Close modal

We asked whether CMLD-2 treatment in activated CD4+ T cells from both type 2 high and non–type 2 high asthmatics would alter their cytokine secretion profiles. As shown in (Fig. 4H and 4I, CMLD-2 significantly inhibited the levels of IL-5 and IL-13 secretion specifically in type 2 high asthmatics. These data are in line with intracellular expression levels of these cytokines (Fig. 4B, 4C). We also observed decreases in IL-17A secretion after CMLD-2 treatment in non–type 2 high asthma (Fig. 4K).

Notably, CMLD-2 has an effect on IFNγ secretion by CD4+ T cells from both non–type 2 high and type 2 high subjects (Fig. 4J). This was unexpected given that, according to our previous data, HuR deletion in mice has no effect on IFNγ production (17).

To investigate putative mechanisms by which CMLD-2 could inhibit cytokine production (specifically in type 2 high asthma), we first determined the steady-state levels of targeted mRNAs in type 2 high and non–type 2 high asthmatics. As shown in (Fig. 5, the steady-state mRNA levels of Th2 cytokines, including IL-4, IL-5, and IL-13 (Fig. 5A–C, respectively), were significantly decreased after treatment with CMLD-2 in type 2 high asthmatics, but with a lower effect on only IL-4 in non–type 2 high compared with the vehicle. Surprisingly, the level of IFNγ was also significantly decreased in the type 2 high, but not non–type 2 high, asthma group (Fig. 5D). The steady-state mRNA levels of GATA3 were also significantly decreased by CMLD-2 treatment in the type 2 high asthma groups (Fig. 5E). Interestingly, the steady-state mRNA levels of RORγt were also significantly decreased by CMLD-2 treatment in both asthma groups (Fig. 5F). HuR mRNA and protein levels were decreased significantly in the type 2 high asthma group and to a lesser extent in the non–type 2 high asthma group (Fig. 5G, 5H).

FIGURE 5.

CMLD-2 treatment results in decreased steady-state mRNA levels of Th2 cytokines, GATA3 and HuR, in type 2 high asthmatics. (AG) Isolated peripheral blood CD4+ T cells from type 2 high and non–type 2 high asthmatics (n = 6–7 in each group) were incubated with CMLD-2 (10 µg/ml) or vehicle for 2 h and then stimulated with anti-CD3/CD28 for 4 d. The steady state of mRNA of proinflammatory cytokines (A–D), GATA3 (E), RORγt (F), and HuR (G) was measured by quantitative PCR. HuR protein levels (H) from purified CD4+ cells were measured by flow cytometry. The values are presented as the ratio of the mRNA target to endogenous control β-Actin for mRNA data (A–G) and percentage of positive cells for HuR protein data (H). Data were analyzed by paired t test comparing CMLD-2 with vehicle within each group of non–type 2 high and type 2 high asthmatics. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 5.

CMLD-2 treatment results in decreased steady-state mRNA levels of Th2 cytokines, GATA3 and HuR, in type 2 high asthmatics. (AG) Isolated peripheral blood CD4+ T cells from type 2 high and non–type 2 high asthmatics (n = 6–7 in each group) were incubated with CMLD-2 (10 µg/ml) or vehicle for 2 h and then stimulated with anti-CD3/CD28 for 4 d. The steady state of mRNA of proinflammatory cytokines (A–D), GATA3 (E), RORγt (F), and HuR (G) was measured by quantitative PCR. HuR protein levels (H) from purified CD4+ cells were measured by flow cytometry. The values are presented as the ratio of the mRNA target to endogenous control β-Actin for mRNA data (A–G) and percentage of positive cells for HuR protein data (H). Data were analyzed by paired t test comparing CMLD-2 with vehicle within each group of non–type 2 high and type 2 high asthmatics. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

GATA3 is the master transcription factor that defines the Th2 cytokine lineage. Because we previously observed reductions in GATA3 mRNA steady-state levels (Fig. 5E) in type 2 high asthmatics, we investigated whether CMLD-2 affects transcript stability using Act D. As shown in (Fig. 6, there were modest but significant reductions in GATA3 mRNA stability with CMLD-2 treatment compared with vehicle (Fig. 6A, 6B). However, there were no differences in RORγτ mRNA stability after CMLD-2 treatment in either group (Fig. 6C, 6D). The Act D data for Th2 cytokines and IFNγ are provided in Supplemental Fig. 3A–H, where Supplemental Fig. 3A–D shows cytokines data on non–type 2 high asthmatics and Supplemental Fig. 3E–H shows the cytokines data on type 2 high asthmatics. As shown in Supplemental Fig. 3, there was no significant difference in any Th2 cytokines (including IL-4, IL-5, and IL-13, except the 1-h treatment for IL-13) or IFNγ mRNA stability with CMLD-2 treatment compared with vehicle in the non–type 2 high asthma group (Supplemental Fig. 3A–D). There were some significant differences in Th2 cytokines mRNA stability with CMLD-2 treatment compared with vehicle in the type 2 high asthma group, including IL-4 (Supplemental Fig. 3E), IL-5 (Supplemental Fig. 3F), and IL-13 (Supplemental Fig. 3G), but not IFNγ (Supplemental Fig. 3H). Taken together, these data suggest the effect of CMLD-2 treatment on stability of Th2 cytokines in type 2 high asthmatics, but not non–type 2 high asthmatics.

FIGURE 6.

CMLD-2 treatment results in decreased GATA3 mRNA stability, but not RORγt, in both non–type 2 high and type 2 high asthmatics. (AD) GATA3 mRNA stability (A and B) and RORγt stability (C and D) after Act D treatment. Isolated peripheral blood CD4+ T cells from non–type 2 high (A and C) and type 2 high (B and D) asthmatics were incubated with CMLD-2 (10 µg/ml) or vehicle for 2 h and then stimulated with anti-CD3/CD28 for 4 d. On day 4, cells were untreated or treated with Act D (3 μg/ml) and harvested at 1, 2, 3, and 4 h. Levels of GATA3 (A and B) and RORγt (C and D) mRNA were determined by quantitative PCR. The values are presented as mean value and derived from three independent experiments, performed in duplicates in separate experiments. Data were analyzed by two-way ANOVA with the Sidak test for comparing vehicle and CMLD-2 values in each time point. *p < 0.05, **p < 0.01, ***p < 0.001. t1/2, half-life.

FIGURE 6.

CMLD-2 treatment results in decreased GATA3 mRNA stability, but not RORγt, in both non–type 2 high and type 2 high asthmatics. (AD) GATA3 mRNA stability (A and B) and RORγt stability (C and D) after Act D treatment. Isolated peripheral blood CD4+ T cells from non–type 2 high (A and C) and type 2 high (B and D) asthmatics were incubated with CMLD-2 (10 µg/ml) or vehicle for 2 h and then stimulated with anti-CD3/CD28 for 4 d. On day 4, cells were untreated or treated with Act D (3 μg/ml) and harvested at 1, 2, 3, and 4 h. Levels of GATA3 (A and B) and RORγt (C and D) mRNA were determined by quantitative PCR. The values are presented as mean value and derived from three independent experiments, performed in duplicates in separate experiments. Data were analyzed by two-way ANOVA with the Sidak test for comparing vehicle and CMLD-2 values in each time point. *p < 0.05, **p < 0.01, ***p < 0.001. t1/2, half-life.

Close modal

The role of Th2 cytokines in asthmatic patients has been extensively investigated over the past few decades, and these investigations have led to the development of new biological therapeutics. Despite such efforts, there are heterogeneous responses to therapy, and many asthma patients continue to have uncontrolled disease. Understanding the molecular heterogeneity of asthma at the posttranscriptional level may aid the field in developing new and personalized treatment modalities. To our knowledge, our study is the first to demonstrate the role of RBP HuR as a critical component of Th2 cytokine signal transduction in asthmatics, especially in the type 2 high endotype.

Initially, we investigated whether there are physiological consequences in HuR KO mice using an OVA model of airway inflammation. We found decreased eosinophils and IL-13 in BALF from HuR KO immunized mice and reduced lung inflammation (Fig. 1). Therefore, we hypothesized that HuR might similarly regulate lung inflammation in human asthma and, more specifically, play an important role in type 2 high asthma. We discovered significantly higher expression of HuR in peripheral CD4+ T cells isolated from type 2 high asthmatics compared with healthy control subjects (Fig. 2). We have shown in the past that HuR plays multiple critical roles in regulating proinflammatory markers in CD4+ T cells by stabilizing target gene mRNAs and increasing translatability in both murine and human systems (17, 1922). In this study, we showed significantly higher expression of Th2 cytokines, including IL-4, IL-5, and IL-13, as well as GATA3 (Fig. 3), in CD4+ T cells from type 2 high asthmatics compared with healthy control subjects after activation. These data from in vitro–activated CD4+ cells are comparable with previous human studies that investigated Th2 cytokine expression in sputum (30) and bronchial biopsies (2) and found significantly higher levels of these cytokines in type 2 high asthmatics compared with healthy control subjects. Intracellular expression of IFNγ in activated CD4+ T cells was similar among type 2 high asthmatics, non–type 2 high asthmatics, and healthy control subjects. We speculate that this may be because of the potential plasticity of the isolated CD4+ T cells into other effector CD4+ T cell types (9, 31, 32). The expression of IFNγ in CD4+ T cells can identify atypical Th2 cells, the skewing and epitope specificity of which are shaped by multiple environmental factors and have been reported in vivo in patients with asthma (9, 31, 32). Plasticity of T cells and/or modulation of Th2 numbers resulting in a shift away from a Th2 to other Th cell subsets have also been reported during allergen immunotherapy (31). The pathological situations where T cells are rendered dysfunctional and incapable of eliminating infected or transformed cells were reported in other studies where the T cells were chronically stimulated (33). Our non–type 2 high asthmatics showed significantly higher expression of IL-17 in their peripheral CD4+ cells compared with the control (Fig. 3), suggesting they may have a mixture of Th2 and Th17 cells or cells that are capable of expressing both cytokines. Previous studies showed IL-4/IL-17 double-positive CD4+ T cells from BAL extracted from asthmatics, which has been linked to the severity of asthma, suggesting a pathogenic role for IL-4hi cells (31, 34).

Few studies are investigating the role of HuR in asthma. Our group and others have shown the regulatory effect of HuR in numerous chronic inflammatory diseases, such as cancer (14, 3538), neuroinflammation (23, 27, 39), liver diseases (40), kidney diseases (41, 42), cardiovascular disorders (43, 44), and skeletal myogenesis (45), which together suggest HuR targeting as a novel and promising therapeutic intervention.

We used two different strategies for interfering with HuR function in our current studies. First, we used the AMPK activator AICAR and then applied the small molecule HuR-specific inhibitor CMLD-2. AICAR (also known as acadesine) has been safely used in multiple human studies (4649); indeed, it is readily available as a dietary supplement. AMPK is a sensitive system that serves as a metabolic sensor and acts like a cellular fuel gauge or a “low fuel warning system” in mammalian cells in response to cellular stresses depleting ATP (5052). AMPK activation during cellular senescence has been linked to reduced HuR function (53). Numerous in vitro and in vivo studies in the field of cancer have used AICAR as a molecular target to inhibit tumorigenesis as prevention and treatment strategies (54, 55).

In this study, we have shown the inhibitory effect of AICAR on expression levels and production of Th2 cytokines (IL-5 and IL-13), as well as GATA3, in both type 2 high and non–type 2 high asthmatics (Supplemental Fig. 2). The IL-4 secretion after treatment with AICAR was unchanged in either group compared with the vehicle control (Supplemental Fig. 2G). We speculate this could be because of the uptake of this cytokine by CD4+ cells in an autocrine fashion during activation (56). Not surprisingly, we have also found an inhibitory effect of AICAR on IFNγ and IL-17 secretion, demonstrating the broader effect of AICAR. This is consistent with several studies that previously showed suppressive effects of AICAR on IFNγ signaling (57), proinflammatory actions of IFNγ (58), and other IFNγ-related pathways, including IFNγ/TNF-α–induced atrophy (59), in different cell types. The inhibitory effect of AICAR on IL-17 production also has been shown in activated murine CD4+ T cells during Th17 polarization (60). Likewise, the inhibitory effect of AICAR on IL-17 and/or IFNγ expression has been seen in activated CD4+ T cells in the lamina propria in colitis (61) and other pathological conditions (62), but no data are available in this respect that we are aware of from patients with asthma. These findings may have relevance to therapeutic modalities to treat non–type 2 high asthma, in which IL-17–secreting cells play important roles. There is a paucity of treatments for this subtype of asthma, because these patients do not optimally respond to corticosteroid therapies, a cornerstone of treating type 2 high asthma.

Finally, we used CMLD-2, a small molecule inhibitor directed against HuR, which inhibits target RNA binding to HuR. CMLD-2 was identified by Wu et al. (63) and has been tested in vitro in multiple cell types (15, 63). We have shown the inhibitory effects of CMLD-2 on cytokine expression and production, as well as in GATA3 expression in type 2 high asthmatics (Fig. 4). Our study is, to the best of our knowledge, the first to demonstrate the inhibitory effect of CMLD-2 on activated CD4+ T cells in asthma. There is limited information on the inhibitory effect of CMLD-2 in other diseases. A few studies using primary or cancerous cell lines showed the inhibitory effect of CMLD-2 on HuR expression, stabilization, proliferation, growth, and apoptosis (6365). Muralidharan et al. (65) have shown the antitumor effects of CMLD-2 on non–small cell lung cancer, showing that CMLD-2 decreases the levels of HuR mRNA and the mRNA of HuR targets in tumor cells. Interestingly, we also found that decreases in both HuR mRNA and protein, as well as HuR targets (Th2 cytokines and GATA3) by CMLD-2, which were predominant in type 2 high asthmatics (Fig. 5). In agreement with these data, the stability of mRNA data of Th2 cytokines (mostly IL-5 and IL-13) was found to be significantly reduced by CMLD2 treatment in the type 2 high, but not in the non–type 2 high, asthma group (Supplemental Fig. 3). This may suggest an increased regulatory effect of HuR on cytokine production in type 2 high asthma. We have also found a modest but statistically significant effect of CMLD-2 on GATA3 mRNA stability, with no difference observed between type 2 high and non–type 2 high asthma (Fig. 6). These reductions, although small, are consistent with what has been previously reported in regard to other HuR target mRNAs (Bcl-2, Msi1, and XIAP) in cancer cell lines (63).

Another interesting finding was the significant inhibitory effect of CMLD-2 on IFNγ secretion levels in both type 2 high and non–type 2 high asthmatics (Fig. 4), suggesting a regulatory effect of HuR on IFNγ expression. Although this disagrees with our previously published data on murine studies in distal lck-Cre HuRfl/fl mice (17), which did not identify IFNγ as a HuR target, this may be because of differences in IFNγ regulation pathways between mice and humans. In contrast, a recent study using OX40-Cre HuRfl/fl mice (generated in our laboratory) showed that HuR ablation can influence IFNγ expression (66). It is our observation that the timing of HuR ablation may affect the results. Distal lck-Cre HuRfl/fl deletes HuR before thymic egress, whereas the OX40-Cre HuRfl/fl model ablates HuR in the periphery after T cell activation. Also, another possibility might be an indirect link between HuR and IFNγ. Our data are in line with one study in human T cells showing posttranscriptional control of IFNγ by HuR, mediated by lymphocyte function–associated Ag 1, and found lymphocyte function–associated Ag 1–mediated mRNA stabilization (67). Further study of the impact of HuR inhibition on IFNγ production in humans, as well as the mechanisms by which these changes are controlled, is warranted.

Unexpectedly, CMLD-2 treatment resulted in decreases in RORγt mRNA steady-state levels (Fig. 5). However, CMLD-2 treatment does not affect RORγt mRNA stability (Fig. 6). Indeed, RORγt 3′ UTR does not have known canonical HuR binding sites. This finding warrants further investigation because it suggests that HuR may directly or indirectly affect RORγt expression.

Also of note is that the inhibitory effect of CMLD-2 on expression levels of HuR-targeted mRNA, as well as RORγt mRNA, was more profound than its effect on the stability of the HuR-targeted mRNA. It is important to note that these significant differences between type 2 high and non–type 2 high asthmatics have been identified using peripheral CD4+ T cells, which are not fully immersed in the milieu of an inflamed airway. This suggests that the T cell programming that these cells have undergone is extensive and long lived. In future studies, it will be important to determine to what extent HuR is upregulated in lung-resident T cells.

Our data suggest that HuR plays a permissive role in both allergen and non–allergen-driven airway inflammation by regulating key genes, and that interfering with its function may be a novel way to treat asthma. This suggests that HuR may be a promising therapeutic target for the alleviation of asthmatic lung inflammation. Our data indicate that HuR plays an important role in CD4+ T cell–mediated inflammation in asthmatics, and that blocking HuR may be effective in decreasing key Th2 and Th17 cytokines produced by peripheral T cells from human asthmatics.

We thank Mohi Shakiba, Marketing and Communications Specialist in the Department of Cell and Developmental Biology, University of Michigan Medical School for the visual abstract.

This work was supported by U.S. Department of Health and Human Services, National Institutes of Health, National Institute of Allergy and Infectious Diseases Grants R01 AI080870 and R21 AI079341.

The online version of this article contains supplemental material.

Abbreviations used in this article

Act D

actinomycin D

AICAR

5-aminoimidazole-4-carboxamide riboside

AMPK

AMP-activated protein kinase

BAL

bronchoalveolar lavage

BALF

bronchoalveolar lavage fluid

KO

knockout

RBP

RNA-binding protein

UTR

untranslated region

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The authors have no financial conflicts of interest.

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