FSTL1 aggravates OVA-induced allergic airway inflammation by activating NLRP3 inflammasome

Asthma is a common respiratory disease characterized by chronic airway inflammation. As a novel inflammatory mediator, follistatin-like protein 1 (FSTL1) can activate immune reaction, suggesting that it may contribute to inflammatory disorders such as asthma. Besides, there are growing evidences that nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) / Interleukin (IL)-1β axis participates in asthma. In this study, we investigated the role of FSTL1 in allergic airway inflammation and its underlying mechanism of activating NLRP3 inflammasome. Methods Circulating FSTL1 and IL-1β levels were quantified in serum of asthmatic patients and controls. Whole-body ablation Fstl1 heterozygous mice (Fstl1 +/- ) and control group were assessed after the experimental treatment. The effects of FSTL1 on NLRP3 inflammasome were also tested in primary macrophages of mice in vitro. The concentration of FSTL1 and IL-1β in serum of asthmatic patients were elevated compared with controls and were positively correlated. FSTL1 deficiency ameliorated infiltration of inflammatory cells,corresponding pathological changes,cytokine responses (IL-1β, IL-5,IL-13), mucous hypersecretion and hyper-responsiveness of airway after Ovalbumin (OVA) exposure in the mouse model. Additionally, inhibition of NLRP3 with MCC950 attenuated FSTL1-induced activation of NLRP3 inflammasome and airway inflammation in vivo and vitro. Conclusions Our data showed that FSTL1 played an important role in allergic airway inflammation by activating NLRP3 inflammasome, providing the possibility that FSTL1 could be applied as a therapeutic strategy on asthma. 4, 8, 12 or 16mg/ml, and were respectively the The results were expressed as changes from


Introduction
Asthma is a common respiratory disease affecting 1-18% of the population in different countries, and is also recognized as the most common chronic disease in children [1].It is a chronic inflammatory PBS overnight at 4˚C. After extensive wash, the sections were incubated with the corresponding HRPconjugated secondary antigens for 1 hour at room temperature and then developed with DAB solution (Boster, China). At last, the slides were counterstained with hematoxylin for about 2 min. The positive area of the target protein was measured by Image-Pro Plus 6.0 software (Media Cybernetics, USA) at ×400 magnification.

ELISA
The levels of IL-4, IL-5, IL-13 IL-1β in BALF supernatant of mice were detected using ELISA kits(CUSABIO, China) according to the manufacturer's instructions. FSTL1 and IL-1β levels in human serum were also measured by ELISA (Abcam, USA). All the calibrations and analyses were performed in duplicate.

Western blot analysis
Prepared lung tissues and PBS washed adherent cells were homogenized by grinding and lysing using ice-cold RIPA buffer in the presence of protease inhibitors. Then, the homogenate was centrifuged at 12000 rpm for 10 min at 4 °C, and soluble supernatants were taken for protein concentration determination by BCA protein assay kit (Boster, China). After that, equal amounts of protein samples (25μg of total protein each) were boiled at 95 °C for 5 min and separated onto SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk for 1 h at room temperature, and then incubated with diluted primary antibodies against NLRP3 (1:1000; CST, MA, USA), pro-caspase1 (1:1,000; Abcam, USA), IL-1β(1:1000; Abcam, USA), GAPDH (1:5000; Abcam, USA) overnight at 4˚C, followed by incubation with HRP-conjugated secondary antibody for 2h at room temperature. The binding of all the antibodies was detected using an enhanced chemiluminescence (ECL) kit (Pierce Biotechnology, USA). All experiments were repeated in triplicate.

Statistical analysis
All data are presented as mean ± standard deviation (mean ± SD). The quantitative analysis of figures of IHC, PAS staining was performed using GraphPad Prism 6 (GraphPad Software Inc, San Diego, CA, USA). The quantitative analysis of IHC was performed using ImageJ. Student's t-test and one-way analysis of variance (ANOVA) were applied to assess differences among groups. Post hoc analysis of SPSS 22.0 software (SPSS Inc., Chicago, IL, USA) was used to evaluate statistical significance. Results were considered statistically significant at p value less than 0.05 (P < 0.05).

1.
Up-regulation of FSTL1 and IL-1β expression in serum of asthmatics.
Characteristics of the asthmatic patients and the controls were described in Table 1, and there are no differences in age and gender between two groups. The levels of circulating FSTL1 and IL-1β in serum were quantified, and they were elevated dramatically in serum from asthma patients compared to the controls (P<0.01) (Fig. 1a, b). In addition, there is a positive correlation between the concentration of FSTL1 and IL-1β in serum (P<0.001) (Fig. 1c ).

FSTL1 + /mice exhibited attenuated infiltration of inflammatory cells and
pathological changes after OVA exposure.
To verify the effect of FSTL1 on airway inflammation in asthma, lung tissues of mice were sectioned , stained with H&E staining, and analyzed for the degree of inflammation. The OVA-sensitized and challenged WT mice exhibited extensive infiltration of inflammatory cells into the peribronchial and perivascular connective tissues areas as compared with the PBS-treated control mice. However, the infiltration was dramatically reduced in FSTL1 +/mice after OVA exposure (Fig. 3a). Besides, the OVAexposed WT group exhibited significant increases in total cell numbers mainly caused by an influx of monocytes and eosinophils when compared to the control group, and these increases were dramatically alleviated in FSTL1 +/mice after OVA exposure( Fig. 4a-e).
3. FSTL1 deficiency reduced goblet cell hyperplasia and mucous hypersecretion in the OVA-induced mice.
PAS and IHC detection with Muc5AC were used to assess the effect of FSTL1 on the mucus production caused by goblet cell hyperplasia in the bronchi. Although many mucus-containing epithelial cells were apparent in the OVA-WT mice when compared to the controls, Strikingly decreasing accumulation of these cells was detected in the OVA-FSTL1 +/mice (Fig. 3b, c).

FSTL1 deficiency inhibited Th cytokine responses in BALF.
After OVA treatment, the secretion levels of IL-1β, IL-4, IL-5, IL-13 were significantly elevated in BALF from WT mice as compared with the control group. And data shown in Figure 4f-i indicated that the levels of IL-1β, IL-5 and IL-13 in BALF from the OVA-FSTL1+/-mice were significantly decreased compared with the OVA-WT mice. The level of IL-4 in BALF from FSTL1 +/mice was also reduced, but comparing with the control group there was no statistical difference.

5.
FSTL1 + /mice showed less airway resistance in response to methacholine compared with WT mice.
The airway responsiveness was assessed within 24 hours after the final challenge. Measurement of lung resistance (RL) showed a significantly increasing in response to methacholine inhalation in the OVA-induced asthmatic mice model, but dramatical reduction of RL induced by OVA was observed in FSTL1 +/mice ( Fig. 2c), demonstrating that FSTL1 may ameliorate OVA-induced AHR.
To determine the effect of FSTL1 on regulating NLRP3 inflammasome in vivo, we examined the expression of the NLRP3 inflammasome components in the lung of mice. As shown in Fig. 5a and 5d , we found dramatically increased NLRP3 immunohistochemical staining in the lung of OVA-WT mice compared with the control group, especially in the alveolar macrophages (AMs), but it was prominently declined in FSTL1 +/mice after OVA exposure. There were no significant differences of pro-caspase 1 detected between FSTL1 +/mice and WT mice after OVA exposure (Fig. 5b, e). But the over-expression of IL-1β induced by OVA exposure was dramatically down-regulated in FSTL1 +/mice compared with WT mice (Fig. 5c, f). Taken together, we suspected that FSTL1 increased the expression of NLRP3, not pro-caspase 1, but it may promote self-cleavage of pro-caspase 1 and then lead to IL-1β production.  7b). We further blocked the effect of FSTL1 with small interfering RNA (siRNA), and the expression of NLRP3, P10 and IL-1β were decreased accordingly as FSTL1 was silenced. These results demonstrated that FSTL1 could activate NLRP3 inflammasome in vitro (Fig. 7c).

MCC950 treatment attenuated FSTL1-induced activation of NLRP3 inflammasome and airway inflammation.
We used a small molecule NLRP3 inhibitor, MCC950, to test whether inflammasome blockade alters FSTL1-induced inflammatory recruitment. Firstly, we tested its blockade action in PMs, the expression of NLRP3,P10 and IL-1β was decreased after MCC950 treatment. We designed animal experiment to take further step to reveal the inflammatory inhibition role of MCC950. After FSTL1 intranasal, the inflammation degree of lung tissues from mice increased compared with the PBS control group.
However, this airway inflammation was significantly inhibited by pre-injection of MCC950.

Discussion
Asthma is a heterogeneous disease, usually characterized by chronic airway inflammation. Limiting the development of inflammatory processes is essential for asthma control, improving the symptoms  Our study showed that there was an obviously positive correlation between FSTL1 and IL-1β in the serum of asthmatic patients. By up-regulating FSTL1 in vitro macrophages, the expressions of NLRP3 and IL-1β were correspondingly increased; after inhibition of NLRP3 with MCC950, FSTL1-induced IL-1β was blocked. FSTL1 stimulation increased NLRP3 and IL-1β expression in biopsies of mice, while the expression was decreased after targeting deletion of FSTL1 in asthmatic model. After intraperitoneal injection of MCC950 in mice, Fstl1-induced airway inflammation was significantly reduced.
However, further work will be needed to illuminate the mechanisms by which FSTL1 is involved in the pathogenesis of asthma. We found FSTL1 promoted the release of inflammatory mediators in asthma by activating NLRP3/IL-1β axis, but it remains unclear whether FSTL1 directly acts on other cells such as T cells, airway epithelial cells and ASM cells, which in turn cause production of a range of inflammatory medium. And the underlying mechanisms need to be identified. Besides, there was no study to look at if differences of FSTL1 levels exist in patients with acute and non-acute exacerbations or in different asthma phenotypes.

Conclusions
Taken together, our results have underscored the key role of FSTL1 in promoting allergic airway inflammation, and that it worked through activating NLRP3 inflammasome. In conclusion, this study and our continuing efforts may provide a novel treatment strategy and/or a diagnostic biomarker for asthma. Correlation was determined by Spearman rank correlation analysis. Data is represented as mean±SD.

Figure 2
The OVA and drug treatment procedures a