MiR-223 plays a protecting role in neutrophilic asthmatic mice through the inhibition of NLRP3 inflammasome

Wide-type (WT) mice (CD45.1⁺C57BL/6 mice, 6-8 weeks) were obtained from the Center for Animal Experiments of Wuhan University (Wuhan, China), and were used for the experiments 1 week after arrival. CD45.1⁺miR-223^−/− mice were purchased from the Jackson Laboratory. All experimental mice were bred in an approved containment facilities with specific pathogen-free food and water under 12 h light/dark cycle. Experiments were approved by the Institutional Animal Ethics Committee of Wuhan University.

The experimental protocol for neutrophilic asthma was performed as previously reported [34]. Mice were sensitized on day 0 with 20 μg of grade V ovalbumin (OVA, Sigma Aldrich, St. Louis, MO, USA) emulsified in 75 ul CFA (Sigma Aldrich) by intraperitoneal (i.p.) injection. On days 21 and 22, all mice were challenged with aerosols consisting of 1% OVA (grade III). Control mice received phosphate-buffered saline (PBS) only. The highly selective NLRP3 blocker, MCC950 (200 mg/kg dissolved in PBS, i.p.) [35] and IL-1β receptor antagonist, anakinra (50 mg/kg dissolved in PBS, i.p.) [36] were given to the OVA/CFA-sensitized miR-223^−/− mice immediately after each challenge, respectively. Control mice were treated with the same volume of PBS for comparison. Mice were sacrificed 24 hours after the final OVA challenge, and then serum, bronchoalveolar lavage fluid (BALF), lungs were collected for subsequent analysis.

MiR-223 agomir is a chemically modified oligonucleotide that can be widely used to upregulate the endogenous expression of miR-223 in animal experiments. Agomirs for miR-223 and the negative control were ordered from RiboBio (Guangzhou, China). The sequence of miR-223 agomir were not provided by RiboBio. miR-223 agomirs (5 nmol in 50ul saline) and negative control agomir were administered intranasally on days 20, 21 and 22 [37, 38]. Control mice were treated with the same volume of saline for comparison.

The tracheas and lungs were lavaged 3 times via a syringe with 0.5 ml PBS containing 0.6 mM EDTA, as previously described [39]. The BALF was centrifuged at 1500 rpm for 7 min at 4 °C and the BALF supernatant was stored at − 80 °C for cytokine analysis. The recovered BALF cells were prepared by cytocentrifugation (TXD3 cytocentrifuge, Xiangyi, Changsha, China) and were stained with Wright-Giemsa (Jiangcheng Bioengineering Institute, Nanjing, China) for differential cell counts (neutrophils, eosinophils, lymphocytes). Four hundred cells were counted for each slide.

The left lung lobe of each animal was resected and fixed in 4% paraformaldehyde buffer for at least 24 h, then dehydrated and embedded in paraffin. Lung sections were cut into 5-um thickness, and were stained with haematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) to assess airway inflammation, goblet cell hyperplasia and mucus secretion at 200× magnification by microscope. Four sections were assessed per lung.

And a scale was used to semi-quantitatively evaluated the severity of peribronchial and perivascular inflammation, as previously described [40]. The extent of mucus production and goblet cell hyperplasia in the airway epithelium was assessed by calculating Apas+/Pbm using Image Pro Plus 6.0 (IPP 6.0) software [41].

Mice were anesthetized with 1% pentobarbital and mechanically ventilated, and AHR was measured by using the animal lung function instrument (Buxco Electronics, Troy, NY, USA), as previously described [42]. Briefly, incremental concentrations of methacholine (ranging from 3.125 to 50 mg/ml) were intratracheally delivered by an attached nebulizer. Baseline airway resistance was assessed using nebulized PBS. Total lung and airway resistance index (RI) were then calculated by the instrument.

Total RNA was isolated from lung tissue using TRIzol (Invitrogen/Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). Complementary DNA (cDNA) synthesis was performed with a miRNA specific primer using Thermo Scientific RevertAid First Strand cDNA Synthesis Kit according to the manufacturer’s manual. Amplification was performed using qPCR with SYBR Premix Ex TaqTM (Takara Bio Inc., Otsu, Japan). All primers were provided by Sangon Biotech (Shanghai, China), and the primers sequences of target genes are presented in the Table 1. The cycle threshold (Ct) of miRNAs were normalized to the Ct of endogenous U6, whereas GAPDH was used to normalize the expression levels of mRNA. The relative gene expression was calculated by the 2^-ΔΔCq method.

To measure the proteins expression of NLRP3, ASC, Caspase-1, IL-1β and IL-18, lung tissues were infiltrated in tissue protein regent with RIPA Lysis Buffer and protease inhibitor (Beyotime Institute of Biotechnology, Haimen, China). The protein concentrations were measured using BCA Protein Assay kit (Thermo Fisher Scientific) following the protocol. The total proteins were separated by 10% SDS-PAGE and then transferred onto a PVDF membrane (Millipore Corp., Billerica, MA, USA). The membranes were blocked with 5% non-fat milk in TBST solution for 2 h at room temperature. The PVDF membranes were subsequently incubated with primary antibodies against NLRP3, ASC, Caspase-1, IL-1β and IL-18 (Abcam, Cambreidge, UK) at 4 °C overnight. After three times washes in TBST for 15 min each, the membranes were incubated with HRP-conjugated secondary antibodies at 37 °C for 2 h. Immunoreactive images were captured with an enhanced chemiluminescence kit according to the manufacturers’ protocol and were detected using the ChemiDocTM Imaging System (Bio-Rad). GAPDH was used as an internal reference.

The levels of IL-4, IL-5, IL-13, interferon gamma (IFN-γ), IL-17A, IL-22, IL-23, IL-1β and IL-18 in BALF were measured using enzyme-linked immunosorbent assay (ELISA) kit (eBioscience, San Diego, CA, USA) according to the manufacturers’ protocols.

To determine the target relationship between miR-223 and NLRP3 mRNA, a luciferase reporter assay was performed using 239 T cells co-transfected with a NLRP3–3’UTR fusion vector and miR-223 mimic, inhibitor and corresponding negative control. Cells were harvested after 48 h, and the luciferase levels were detected using a Dual-Luciferase Reporter Assay System (Promega) according to the manufacturers’ instructions.

To investigate the role of specific miRNAs in the OVA-induced neutrophilic asthmatic mice model (Fig. 1.a), we identified that the miR-223 expression level in the lungs of the OVA-induced mice model (23 day) was significantly upregulated compared with the PBS –induced mice (Fig. 1.b). In addition, whereas the expression of miR-223 was increased after sensitization (1 day) and after the first challenge (22 day), there was no significant difference compared with the control group (Fig. 1.b). These evidences provided a basis to assess the biological function of miR-223 during neutrophilic asthma.

To further elucidate the role of miR-223 in the regulation of neutrophilic inflammation in asthma, we used miR-223^−/− mice sensitized and exposed to OVA, followed nebulizing 1%OVA for 2 consecutive days after 3 weeks. MiR-223^−/− mice exposed to OVA had dramatically increased numbers of infiltrating inflammatory cells and mucus hypersecretion, as determined by the number of PAS staining cells in airways, when compared with OVA-challenged WT mice (Fig. 1.c-e). No inflammatory cells and PAS-positive cells were found in either WT mice or miR-223^−/− mice that were exposed to PBS. Furthermore, miR-223^−/− mice exposed to OVA had markedly increased the number of total inflammatory cells, neutrophils, eosinophils and lymphocytes in BALF when compared to OVA-challenged WT mice (Fig. 1.f). In addition, the airway resistance of mice to increasing concentrations of methacholine was detected. The results demonstrated that RI was significantly difference at higher concentrations of methacholine in OVA-challenged miR-223^−/− mice compared with OVA-challenged WT mice (Fig. 1.g). In comparison with the OVA-challenged WT mice, OVA-challenged miR-223^−/− mice showed a significant reduction in the compliance of lung at the concentration of 3.125 and 25 mg/ml. Subsequently, the expression of Th1-related cytokines (IFN-γ), Th2-related cytokines (IL-4, IL-5, IL-13) and Th17-related cytokines (IL-17A, IL-22, IL-23) in BALF were examined by ELISA, respectively. IL-4, IL-5, IL-13, IL-17A, IL-22, IL-23 levels were markedly raised in BALF of OVA-challenged miR-223^−/− mice compared with OVA-challenged WT mice (Fig. 1.h-m). However, IFN-γ release was not significantly altered in OVA-challenged miR-223^−/− mice compared with OVA-challenged WT mice (Fig. 1. n). Other cytokines including IL-1β, IL-18 were also detected in BALF. Notably, IL-1β release dramatically increased in miR-223^−/− mice compared with OVA-challenged WT mice (Fig. 1. o). Similarly, the expression of IL-18 was also increased in OVA-induced miR-223^−/− mice, but the difference was not significant as IL-1β (Fig. 1. P). Thus, these results reveal that miR-223 is associated with airway inflammation, mucus production, AHR, the Th2 and Th17 responses and pro-inflammatory cytokines release in the lung tissues of neutrophilic asthmatic mice.

Based on the finding that miR-223 regulates airway inflammation and strengthen IL-1β release, we next explored the role for miR-223 target mRNA, NLRP3. To further confirm the effects of miR-223 deficiency on NLRP3 inflammasome, we detected the protein expression of NLRP3 in lung tissues by western blotting. Moreover, the pulmonary expression levels of NLRP3 protein were significantly increased in OVA-challenged miR-223^−/− mice compared with OVA-challenged WT mice (Fig. 2.a). Consistent with this result, the expression levels of pro-caspase-1 (45KD) and cleaved caspase-1 (20KD) and IL-1β and IL-18 were also significantly increased. However, miR-223 deficiency did not alter the protein expression of ASC, pro-IL-1β, pro-IL-18 (Fig. 2.a-b). In accordance with these results above, we observed the mRNA expression of NLRP3, Caspase-1, IL-1β were markedly higher in lung tissue of OVA-challenged miR-223^−/− mice compared with OVA-challenged WT mice (Fig. 2.c). These data indicate that miR-223 plays a selective and functional role in regulating a critical component of NLRP3 inflammasome.

Next we verified whether blocked either the NLRP3 inflammasome or IL-1β could directly further attenuate airway inflammation in miR-223^−/− mice. On days 21 and 22 after sensitization, miR-223^−/− mice were treated with NLRP3 inhibitor (MCC950) or IL-1 receptor antagonist (anakinra) immediately after each OVA challenge, respectively. Both treatment dramatically decreased the numbers of infiltrating inflammatory cells and mucus hypersecretion in the lung, as well as the number of total inflammatory cells, neutrophils, eosinophils and lymphocytes in BALF (Fig. 3.a-d). Similarly, airway resistances were significantly abolished by both MCC950 and anakinra administration (Fig. 3.e). Although inflammatory cytokines IFN-γ was unaltered, the levels of IL-4, IL-5, IL-13, IL-17A, IL-22, IL-23, IL-1β and IL-18 were significantly lower after treatment (Fig. 3.f-n). To confirm the role of MCC950, the protein levels of NLRP3 and its important components, ASC and caspase-1 and the typical downstream proteins, IL-1β and IL-18 were detected by western blotting. As shown in Fig. 4.a-c, MCC950 administration nearly abolished the protein expression of caspase-1, although it did not alter the protein expression of ASC. Consistent with this, the protein expression of IL-1β was remarkably decreased by treatment with MCC950, and IL-18 also decreased after treatment. On the other hand, the protein expression of pro-IL-1β, pro-IL-18 were unaltered. Thus, enhanced NLRP3 activation followed by mainly increased mature IL-1β resulted in an enhanced airway inflammation in miR-223^−/− mice.

To further verify the interaction between NLRP3 and miR-223, bioinformatics analysis was performed to predict the presence of binding sites of miR-223 and the NLRP3 3′-UTR. Similar to previous publications [23], complementary sequences between miR-223 and the NLRP3 3′-UTR was also observed. And dual luciferase reporter assay also showed that the overexpression of miR-223 diminished the luciferase activity of the NLRP3 3’UTR of the wild-type, while the effect was abolished with the mutant NLRP3 3′-UTR. Conversely, inhibition of miR-223 enhanced the luciferase activity with the NLRP3 3’UTR of the wild-type, but not that of the mutant NLRP3 3′-UTR (unpublished data). Collectively, these findings confirmed that NLRP3 was a direct target of miR-223.

To explore the effect of miR-223 on airway inflammation in vivo, miR-223 agomirs was delivered intranasally to mice 6 h before OVA-challenge (Fig. 1.a). As shown in Fig. 5.a, the expression of miR-223 in lungs after transfection agomirs was increased fivefold compared with the negative control. Notably, treatment with miR-223 agomirs significantly resulted in a reduction of infiltrating inflammatory cells and mucus hypersecretion in lungs compared with the negative control group (Fig. 5.b-d). In comparison with the negative control group, the inflammatory cells including neutrophils, eosinophils and lymphocytes in BALF were remarkably reduced in miR-223 agomirs treatment group (Fig. 5.e). Besides, remarkable differences in the airway resistances were observed between miR-223 agomirs treatment group and negative control group (Fig. 5.f). Compared with negative control group, miR-223 agomirs treatment effectively reduced the expression of IL-4, IL-5, IL-13, IL-17A, IL-22, IL-23, IL-1β and IL-18 in BALF, while there was no difference in IFN-γ expression between the two groups (Fig. 5.g-o). Furthermore, miR-223 agomirs treatment inhibited the expression of NLRP3 at the level of mRNA and the protein. This was accompanied with the repression of caspase-1and IL-1β protein and mRNA expression (Fig. 5.p-r). Overall, these results confirm that miR-223 play a critical role in regulating airway inflammation and represent a basic study for the application of miRNA in the treatment of asthma.