Background
Our understanding of asthma immunopathogenesis has been advanced over the last decade [
1]. Much evidence has pointed to the importance of airway epithelium in asthma inception [
2]. Airway epithelium, as an immunological barrier, is thought to initiate and orchestrate airway inflammatory responses to environmental triggers through multiple mechanisms, one of which is airway-expressing Toll-like receptors (TLRs) pathway [
3]. TLRs are important for early recognition of the inhaled environmental antigens, further triggering innate and adaptive immunity [
3]. This process is tightly regulated by intracellular members of the IL-1 receptor-associated kinase (IRAK) family. IRAK-M, an inactive IRAK isoform, functions as a negative regulator of NF-κB signaling pathway dependent on the adaptor protein Myeloid Differentiation factor (MyD) 88 coupled with TLRs [
4]. Interestingly, a recent study demonstrated that IRAK-M activation in Th2 airway inflammation induced by IL-33 was dependent on the prolyl cis–trans isomerase PIN1 mechanism [
5].
Genetic studies have linked IRAK-M SNP variants to both early- and late-onset asthma, suggesting that airway-expressing IRAK-M may be involved in inappropriate activation of innate immunity in the bronchial cells [
6,
7]. Aberrant expression of IRAK-M in airway epithelium has displayed the opposing role, anti- or pro-inflammatory effect, in mediating lung injury in the different context of the pathophysiological settings [
8‐
11]. For example, IRAK-M expression was shown to be significantly elevated in the airway epithelial cells and mucosa of the experimental murine asthma models and asthmatic patients [
7,
10,
12]. Our previous investigations reported that IRAK-M knockout exacerbated allergic airway inflammation via activation of macrophages and dendritic cells and promotion of Th2 immunity in the mouse models induced by OVA or house dust mite (HDM) [
12,
13]. On the other hand, overexpression of IRAK-M showed proinflammatory effects. For instance, Wu et al. reported that persistent expression of IRAK-M induced by a Th2 cytokine IL-13 in asthmatic airway epithelial cells inhibited epithelial TLR2 defense mechanism [
10]. Consistently, we used an IRAK-M knockout mouse model chronically exposed to OVA stimulation and showed that IRAK-M maintained Th2 airway inflammation and inhibited the DC-mediated Th1 activation [
7]. Taken together, current evident supported IRAK-M involvement in the pathophysiology of lung inflammation mainly through activation of macrophages and dendritic cells or impact on T cell differentiation.
Given the multiple lines of evidence indicating that IRAK-M is involved in modulation of innate immunity, whether IRAK-M regulates airway epithelial inflammation and downstream signaling pathways has not been well understood. Here we performed in vitro investigations to test this possibility by examining the effect of IRAK-M knockdown on cellular immunity in immortalized lung epithelial cell lines under inflammatory stimulations. We found that IRAK-M showed effects on inflammatory events of lung epithelial cells, with an impact on Th1 cytokine CXCL10 production possibly through JNK and p38 MAPK pathways.
Methods
Cell culture
Human airway epithelial cell line BEAS-2B cells and human alveolar epithelial cell line A549 cells were purchased from Shanghai Institute of Life Sciences, Chinese Academy of Sciences (Shanghai, China). BEAS-2B cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) media (HyClone, USA) and A549 cells were cultured in F-12K media (Gibco, USA). All media were supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (HyClone). All cells were cultured at 37 °C in a humidified incubator of 5% CO2.
IRAK-M siRNA (si-IRAK-M; sense 5′-CCU-AAC-AUA-UGC-UGU-CAA-ATT-3′; antisense 5′-UUU-GAC-AGC-AUA-UGU-UAG-GTT-3′) were obtained from GenePharma (Suzhou, Jiangsu, China). Nontargeting pool-negative siRNA (si-con; sense 5′-UUC-UCC-GAA-CGU-GUC-ACG-UTT-3′; antisense 5′-ACG-UGA-CAC-GUU-CGG-AGA-ATT-3′) were used as controls. siRNAs transfections of BEAS-2B cells and A549 cells were performed using GP-transfect-Mate transfection reagents. siRNAs and transfection reagents were mixed following manufacturer’s protocol and incubated with the cells for 48 h. Cells were harvested with TRIzol reagent (Thermo Fisher Scientific, USA) for quantitative real-time polymerase chain reaction (qRT-PCR) or harvested with protein lysis buffer (5 ml 20% SDS, 0.77 g DTT (1 M), 3 ml Tris–HCl (1 M, pH 6.8), 5 ml 100% glycerol, 32 ml H2O) freshly added 1 mM phosphatase and protease inhibitor (Roche, Switzerland) for western blot.
Airway epithelial cell model of inflammation
IL-1β (1 ng/ml; R&D system, USA), or TNF-α (10 ng/ml; R&D system), or IL-33 (100 ng/ml; R&D system), or HDM (10 μg/ml; Cosmo Bio USA, USA) was separately added to BEAS-2B or A549 cells. Cells were harvested at 0, 6, 12, 24 h for Western blot. BEAS-2B and A549 cells were starved in serum-free media for 24 h after IRAK-M siRNA transfection. The cell-free supernatant and cells were collected at 24 h after stimulation with IL-1β, or TNF-α, or IL-33, or HDM for analysis of cytokines. To reflect the effect of IRAK-M on the JNK and p38 MAPK signaling pathways in airway epithelial cells, BEAS-2B and A549 cells were harvested at 0, 10, 30, 60 min for Western blot after IRAK-M siRNA transfection. Cells were starved and stimulated. BEAS-2B and A549 cells were incubated with JNK inhibitor SP600125 (20 μM; MedChemExpress, USA) or p38 MAPK inhibitor SB203580 (10 μM; MedChemExpress) for 2 h. After 24 h of stimulation, cell-free supernatant and cells were harvested for cytokines analysis.
Western blot
Whole-cell protein extracts were separated on 10% SDS–polyacrylamide gel electrophoresis gels and transferred to nitrocellulose filter membranes. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 h. Then the membrane was cut and incubated with the respective primary Abs at 4 °C overnight. The Abs were as follows: anti-mouse IRAK-M, anti-mouse JNK, anti-mouse Phospho-JNK, anti-mouse p38 MAPK, anti-mouse Phospho-p38 MAPK and anti-mouse GAPDH (Cell Signaling Technology, USA). Primary Ab application was followed by incubation with HRP-conjugated secondary Abs (rabbit anti-mouse IgG) (Cell Signaling Technology) at room temperature for 1 h. After washing for three times with TBS-T, membranes were scanned using Odyssey CLx imaging system (LICOR, USA). Semi-quantitation was analyzed with Image J software (National Institutes of Health). Protein expression levels were normalized to GAPDH.
ELISA
Commercial ELISA kits were used to measure the concentrations of the cytokines, including IL-6 (R&D system), IL-8 (R&D system), CXCL10 (R&D system and Laizee biotech, Shanghai, China), CXCL11 (R&D system), and IFN-γ (R&D system), in supernatants collected from cells.
Quantitative real-time PCR
qRT-PCR was used to reflect mRNA expression of IRAK-M and cytokine and chemokines, including IL-6, IL-8, CXCL10, CXCL11 and IFN-γ. Total RNA prepared from the BEAS-2B or A549cells using TRIzol reagent was reverse transcribed into cDNA using the ALL-IN-ONE RT MasterMix (Applied Biological Materials, Canada). qRT-PCR was performed on the CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, USA) in a 20 μl of final reaction volume containing 1 μl of cDNA and qPCR MasterMix (Applied Biological Materials). The relative quantities of mRNAs were calculated with the comparative cycle threshold method and normalized using human GAPDH as an internal control. The primer sequences are shown in Additional file
1: Table S1, as supplementary data.
Participants
A total of 137 individual patients with asthma was recruited from Asthma Clinic of Beijing Aviation General Hospital between September 2021 and March 2022. Asthma was diagnosed by the respiratory specialists. All patients were fulfilled the diagnostic criteria of the Global Strategy for Asthma Management and Prevention [
14]. The study was approved by the Ethics Committee for Human Research of Beijing Aviation General Hospital (MHZYY 2014-05-01). All participants provided informed written consent at enrolment.
Genotyping
Genomic DNA was extracted from peripheral blood leukocytes by using TIANamp Genomic DNA Kit (TIANGEN BIOTECH, Beijing, China) according to manufacture protocol. Two SNPs of IRAK-M (rs1624395, forward 5′-ATGGGATTGGGAGAGAAGCC-3′ and reverse 5′- TAAGCCAAAAGCCAGGTCCA-3′; rs1370128, forward 5′-ACCATGGGGTCTGCATCATTT-3′ and reverse 5′-CTGGTTGGTTCTCCTGCAAC-3′) were genotyped by SNaPshot. The PCR products were sequenced on an ABI 3730xl DNA Analyzer (Applied Biosystems) and analyzed using the GeneMapper 4 software. The genotyping efficiency for IRAK-M SNP was > 95% and minor allele frequency was > 5%.
Measurement of serum CXCL10 in asthma patients
Fasting blood samples were harvested from patients with asthma in the morning and centrifuged at 12,000 rpm. Serum was stored at − 80 °C until measurement. Serum CXCL10 concentrations were measured using a commercial ELISA kit (R&D system) following the manufacturer’s instructions.
Statistical analysis
Data were expressed as mean ± SEM. Multiple comparisons were performed by one-way ANOVA with Tukey post-hoc test. Comparisons between two groups were made by unpaired Student t-test (GraphPad Prism Version 9.0, GraphPad, San Diego, CA). P-value < 0.05 was considered significant.
Discussion
One of important functions of airway epithelium is to maintain airway homeostasis, which is balanced with potentially deleterious inflammatory effects [
2]. Many of identified asthma susceptibility genes, including IRAK-M, are expressed by airway epithelial cells, and dysregulated airway epithelial function is thought to be critical for controlling disease induction [
6,
15]. Upon stimulation with aeroallergens, airway epithelial cells, via TLRs (including TLR 2 and 4), release the early epithelial-derived cytokine and chemokines leading to the activation of innate immunity that mediate the type 2 inflammatory responses in the airways [
16,
17].
The expression of human IRAK-M of TLR signal pathway is not limited to monocytes, macrophages, but also found in airway epithelial cells and alveolar epithelial cells [
6]. Current literature points out the importance of IRAK-M in regulation of epithelial innate immunity [
10]. Significant induction of IRAK-M expression was observed in airway epithelial cells during asthmatic process [
5,
7,
10,
12,
13]. In this study, using an airway epithelial BEAS-2B cell line and alveolar malignancy-derived A549 cells line, we investigated the effect of IRAK-M with RNA interference on inflammatory response in lung epithelial cells. We exposed the cells to the following typical stimuli: (1) the early response proinflammatory cytokines IL-1β and TNF-α; (2) an epithelial-released cytokine IL-33 that induced airway inflammation via IRAK-M [
5]; (3) an environmental aeroallergen HDM triggering allergic inflammatory response via airway-epithelium expressing TLR [
16]. We found that individual exposure to IL-1β, or TNF-α, or IL-33, or HDM significantly induced IRAK-M expression at mRNA and protein levels in both BEAS-2B and A549 cells in a time-dependent manner.
Airway epithelial cells play an instructive role in shaping innate or adaptive immune responses during asthmatic process by releasing multiple chemokines and cytokines [
1]. By silencing IRAK-M expression in BEAS-2B and A549 cells, we demonstrated IRAK-M knockdown increased the production of proinflammatory cytokines and chemokines, including IL-6 and IL-8, CXCL10 and CXCL11, at both mRNA and protein levels, in response to a variety of stimuli (including IL-1β, TNF-α, IL-33, and HDM). IL-6 and IL-8 are two classic cytokines that are induced by NF-κB with important roles in innate immunity. Systemic elevation of IL-6 was seen in individuals with an exacerbation-prone asthma [
18] and increased production of IL-8 by bronchial epithelium cells involved in neutrophilic inflammation in asthmatic process [
19].
In this study, to the best of our knowledge, we first reported that IRAK-M regulated expression of chemokine receptor CXCR3 ligands CXCL10 and CXCL11, not CXCL9, by airway epithelial cells upon inflammatory stimulation. CXCL10, the most studied in airway inflammation [
20], is a major chemokine produced by bronchial cells in response to several pathogens, such as RV, respiratory syncytial virus and HDM allergen [
21‐
23]. Activated airway epithelial cells are the major source of CXCL10 in the lungs [
24], which has been shown to be involved in both bronchial inflammation and airway hyper-responsiveness in a mouse model of asthma [
25].
CXCL10 is thought to be induced by Th1 cytokine IFN-γ in a variety of cells that promotes Th1-biased airway inflammation [
21]. However, our findings indicated that CXCL10 production by lung epithelial cells was not concomitant with IFN-γ elevation, supporting the previous study showing CXCL10 expression is partially independent of IFN-γ in an OVA-challenged asthma mouse model [
25,
26]. A previous study has shown that IRAK-M deficiency leads to excessive activation of p38 MAPK and NF-κB pathways [
27], which is consistent with our observation showing overexpression of p-IκBα, p-JNK, and p-p38 MAPK in stimulated IRAK-M KO dendritic cells (DCs) [
12]. Moreover, bone marrow monocytes (BMMs) deficient in IRAK-M treated with a toll-like receptor 1/2 (TLR1/2) agonist Pam3Csk increased phosphorylation of p38 MAPK [
28]. We found that IRAK-M silencing partly, but significantly, upregulated activation of JNK and p38 MAPK pathways in BEAS-2B and A549 cells after several inflammatory stimuli. Our data was supported by the previous investigation demonstrating p38 MAPK inhibitor SB203580 inhibited the activity but not the activation of p38 MAPK [
29]. The effect of IRAK-M on CXCL10 expression was counteracted by JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 incubation. Our present study suggested alternative pathways that involve the CXCL10 upregulation in lung epithelial cells. Therefore, we inferred that upon stimulation, IRAK-M might involve CXCL10 secretion by lung epithelial cells partly through alternative JNK and p38 MAPK pathways. Our data supported the previous reports showing that significantly less release of CXCL10 from BEAS-2B-eosinophil coculture by blockade of p38 MAPK with its specific inhibitor [
30]. These results might indicate a novel pathway that explains the regulatory mechanism of lung epithelial production of CXCL10 in initiation and progression of asthma independent of IFN-γ.
Asthma pathogenesis is caused by gene and environment interaction. To further support this notion, a meta-analysis of pathway enrichment showed that many of the dysregulated genes expressed by nasal or epithelia were linked to asthma pathogenesis [
15]. Our previous study performed in a Chinese population had identified two asthma susceptible SNPs, rs1624395 and rs1370128, in IRAK-M locus that were associated with mRNA expression of IRAK-M by circulating monocytes [
7]. In this study, we further provided evidence that CXCL10 secretion by lung epithelial cells was partly associated with IRAK-M silencing upon the inflammatory stimulation. Moreover, we found IRAK-M SNPs rs1624395 and rs1370128 both influenced the serum concentrations of CXCL10 in patients with adult asthma. Asthma patients carrying homozygote G/G of rs1624395 had a significantly higher level of serum CXCL10 than those carrying homozygote A/A. CXCR3 chemokines (including CXCL10) secretion has been shown to be regulated by activation of TLRs [
31]. IRAK-M negatively regulates TLR signaling [
4], thus, it was reasonable that IRAK-M silencing increased CXCL10 expression as presented by our data. These findings implicate that IRAK-M SNPs have an important role in influencing CXCL10 production in asthma patients that should be further investigated in future study.
There are some limitations in our study. First, our study focused only on BEAS-2B and A549 cells as an in vitro model because the scope of our experiments needs cell lines that grow robustly and there is a substantial literature underpinning BEAS-2B and A549 cells use in inflammation research [
8,
32,
33]. Because BEAS-2B and A549 cells are both cell lines, our in vitro results might not completely reflect the real status of bronchial epithelial cells in vivo. Differences in IRAK-M expression between BEAS-2B cells and A549 cells could lead to inconsistent results after stimulation between the two cell lines. Samples from bronchial epithelial cells of asthma patients may represent the real effect of IRAK-M on airway inflammatory responses. Secondly, short-term exposure of inflammatory stimuli might not produce chronic inflammatory response in airway epithelial cells. In addition, we only measured the serum concentration of CXCL10 in asthma patients, the most studied and important for asthma pathogenesis among three CXCR3 chemokines [
21,
22]. A previous study performed children asthma patients showed the associations between higher serum CXCL10 levels and disease exacerbation and severity [
34]. We did observe allele G carriers had a higher level of serum CXCL10, however, we were unable to present the underlying mechanism how IRAK-M SNPs influence the serum level of CXCL10 in this study. Future studies are needed to comprehensively evaluate the expressional changes in the samples, including airway epithelia, sputum, and bronchoalveolar lavage fluid, from asthma patients in the context of genetic architecture.
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