Nuclear receptor corepressor 1 deficiency exacerbates asthma by modulating macrophage polarization

Macrophage polarization plays an important role in asthma. Nuclear receptor corepressor 1 (NCOR1) plays an important role in metabolic and cardiovascular diseases by regulating the function of macrophages. The aim of this research was to examine the role and mechanism of macrophage NCOR1 in the development of asthma. We used ovalbumin (OVA) to induce macrophage NCOR1-deficient mice for asthma formation. Our results revealed that macrophage NCOR1 deficiency markedly enhanced allergic airway inflammation. In addition, NCOR1 deficiency in macrophages was found to enhance M2 polarization. Mechanistic studies suggested that NCOR1 promoted macrophage polarization by interacting with PPARγ, contributing to the pathogenesis of asthma. In conclusion, macrophage NCOR1 deficiency promoted the regulation of M2 programming by enhancing PPARγ expression to exacerbate asthma. Macrophage NCOR1 might be a potential target for the treatment of asthma.


INTRODUCTION
Asthma is characterized by chronic airway inflammation and reversible airway obstruction affecting ~334 million people worldwide [1].Over the past few decades, the incidence of asthma has increased, resulting in a huge global health and economic burden [2].Clinical medications for asthma, such as inhaled corticosteroids, do not change the natural history of asthma [3].Therefore, more in-depth research on asthma is imperative.
Although the pathogenesis of asthma is still poorly understood, studies have indicated that genetic, environmental, infectious, and nutritional factors impact the emergence of asthma [4].Notably, most patients with asthma have the hallmark of Th2 inflammation, which is associated with certain cytokines (IL-2, IL-5, and IL-14) and inflammatory cells (eosinophils, mast cells and macrophages) [5,6].Among them, macrophages, as the most enriched immune cells in the lung, show extremely high plasticity according to changes in the lung microenvironment.In general, macrophages can differentiate into the M1 phenotype (classically activated macrophages) when stimulated by interferon-γ (IFN-γ) or tumor necrosis factor-α (TNF-α) [7].On the other hand, macrophages can differentiate toward the M2 phenotype (alternatively activated macrophages) after IL-4 or IL-13 treatment [8,9].In asthma, M2 macrophages infiltrate the airways of patients during disease progression and mediate type 2 immune responses [10].At the same time, Th2-associated factors can further exacerbate macro-phage differentiation toward the M2 phenotype, leading to a vicious cycle of asthma [11][12][13].However, the exact mechanism of macrophage differentiation in asthma is still under investigation.
Nuclear receptor corepressor 1 (NCOR1) is a central regulator of transcription that inhibits the transcriptional activity of transcription factors by binding histone deacetylases to their corresponding sites [14].These transcription factors include peroxisome proliferator-activated receptor (PPAR), estrogen-related receptors, and liver X receptor β (LXRβ) [15][16][17].For example, NCOR1 interacts with LXRβ in microglia, and knockdown of NCOR1 in macrophages protects against ischemic stroke [18].Deficiency of Ncor1 in macrophages exacerbates periodontitis by regulating Cebpα transcription through PPARγ, promoting osteoclastogenesis and neutrophil accumulation in mice with experimental periodontitis [19].In a previous report, macrophage NCOR1 has been shown to prevent atherosclerosis via activation of PPARγ [15].It has also been reported that M1-related genes are downregulated, while the expression of M2-type genes is upregulated in macrophage NCOR1 knockout (MNKO) mice [20].Thus, macrophage NCOR1 may play a key role in asthma.
Therefore, we intended to investigate the involvement of macrophage NCOR1 in asthma.MNKO mice were used to investigate the effect of macrophage NCOR1 deletion on inflammation and macrophage differentiation in asthma.Finally, we investigated the cellular mechanism by which NCOR1 regulates macrophage differentiation in vitro.

Macrophage NCOR1 deficiency exacerbates asthma in mice
In a mouse model of asthma induced by OVA peptides (Fig. 1A), the protein level of NCOR1 was significantly decreased in the lung tissues of asthmatic mice compared with control mice (Fig. 1B, C).Moreover, immunofluorescence staining illustrated that NCOR1 was markedly decreased in macrophages in the bronchoalveolar lavage fluid (BALF) of asthmatic mice (Fig. 1D, E).These results indicated an essential role of macrophage NCOR1 in the pathogenesis of asthma.To further investigate the role of macrophage NCOR1 during allergic airway inflammation, we generated MNKO mice by using the cre-lox system.RT-QPCR showed that NCOR1 mRNA levels were almost undetectable in bone marrow-derived macrophages (BMDMs) isolated from MNKO mice than LC mice (Fig. 1F) [20].Hematoxylin and eosin (HE) staining showed that OVA-induced infiltration of inflammatory cells around the bronchovascular bundle was significantly higher in MNKO mice than in LC mice (Fig. 1G, H).Similarly, Masson's trichrome staining demonstrated that OVA-induced peribronchial collagen deposition was significantly higher in MNKO mice (Fig. 1I, J).
We further used periodic acid-Schiff staining (PAS) to examine mucus production.Severe goblet cell hyperplasia and mucus hypersecretion were observed in OVA-challenged mice, and OVAchallenged MNKO mice had a significantly more severe phenotype compared with LC mice (Fig. 2A, B).In addition, we measured the mucus integral component MUC5AC by immunohistochemistry. Consistently, immunohistochemistry analysis demonstrated that asthmatic MNKO mice had a remarkable increase in the number of cells expressing MUC5AC compared with asthmatic LC mice, suggesting improved goblet-cell metaplasia after asthma (Fig. 2C, D).Collectively, these data suggested that macrophage NCOR1 deficiency exacerbated asthma in mice.

Macrophage NCOR1 deficiency exacerbates Th2 inflammation in mouse asthma
Next, we compared inflammatory cell subtypes and cytokines in the mouse BALF.Giemsa staining demonstrated that OVA induced significantly more eosinophils and lymphocytes and fewer macrophages in the BALF of MNKO mice than in that of LC mice (Fig. 3A, B).For the reason for the decrease in macrophages in BALF, we believed that NCOR1 deficiency in macrophages may affect macrophage proliferation [20].More importantly, OVAchallenged MNKO mice showed significantly increased BALF total cells compared with OVA-challenged LC mice (Fig. 3C).Analysis of cytokines in BALF by ELISA revealed significantly higher levels of Th2 cytokines such as eotaxin1 and IL-13 in asthmatic MNKO mice than in asthmatic LC mice (Fig. 3D, E).The level of IL-4, another Th2 cytokine, showed an increasing trend in the BALF of asthmatic MNKO mice (Fig. 3F).Collectively, these results demonstrated that  Macrophage NCOR1 deficiency enhances M2 polarization Importantly, M2 macrophages play an important role in allergic asthma [21].Indeed, the protein level of Arginase1, a signature for M2 macrophages, was increased in lungs from OVA-challenged LC mice compared with saline-treated LC mice (Fig. 4A, B).Therefore, we examined the effect of NCOR1 on macrophage M2 activation.Western blotting showed that the protein level of Arginase 1 was dramatically increased in asthmatic MNKO mice compared with asthmatic LC mice (Fig. 4A, B).Likewise, RT-QPCR analysis of IL-4, Ym1, Fizz1 and Arginase 1 showed consistent results (Fig. 4C-F).These results suggested that macrophage NCOR1 deficiency improved the induction of M2 macrophages.To evaluate the effect of MNKO on macrophage differentiation, we treated LC and MNKO BMDMs with IL-4 and IL-13 or LPS.The protein level of Arginase 1 was significantly increased in MNKO BMDMs compared with LC BMDMs after IL-4 and IL-13 stimulation (Fig. 4G, H), and similar results were obtained by RT-QPCR analysis of YM1 and Fizz1 (Fig. 4I, J).In addition, we also observed the effect of MNKO on M1 differentiation by stimulating BMDMs with LPS.RT-QPCR analysis revealed that after adding LPS, NCOR1 knockout in macrophages strongly inhibited LPS-induced expression of M1 macrophage genes (Fig. 4K, L).Overall, these data suggested that macrophage NCOR1 deficiency enhanced M2 polarization and inhibited M1 polarization.

PPARγ signaling mediates the effects of NCOR1 deficiency on M2 polarization
An intact IL-4/IL-13/PPARγ axis is critical for the maturation of macrophage M2 activation, and NCOR1 deficiency in macrophages leads to an alternative macrophage polarization phenotype upon PPARγ stimulation [15,22].Therefore, we used western  blotting to detect the protein level of in LC and MNKO BMDMs after IL-4 and IL-13 stimulation.Remarkably, IL-4 and IL-13 stimulation induced a significantly increased protein level of PPARγ in MNKO BMDMs compared with LC BMDMs in a timedependent manner, indicating that NCOR1 deficiency enhanced PPARγ expression (Fig. 5A, B).To further demonstrate that PPARγ played a key role in macrophage M2 polarization in the absence of NCOR1, we treated LC and MNKO BMDMs with the PPARγ inhibitor T0070907 or Pparγ siRNA.Western blotting analysis showed that the protein level of PPARγ and Arginase 1 was significantly lower in MNKO BMDMs than in LC BMDMs after T0070907 treatment (Fig. 5C-E).Consistently, Western blotting and RT-QPCR analysis revealed that Pparγ siRNA efficiently repressed gene expression and the protein level of PPARγ in both LC and MNKO BMDMs (Fig. 5F, G, I).Accordingly, Pparγ siRNA notably decreased Arginase 1 gene expression and protein level in MNKO BMDMs compared with LC BMDMs (Fig. 5F, H, J).Collectively, these findings supported that macrophage NCOR1 deficiency promoted macrophage M2 polarization by enhancing PPARγ expression.

DISCUSSION
Although NCOR1 exerts a clear and important function in the pathogenesis of atherosclerosis and myocardial infarction [15,20], it is unclear how macrophage NCOR1 affects asthma.In the present investigation, we demonstrated that NCOR1 deficiency in macrophages aggravated OVA-induced asthma.Mechanistically, NCOR1 deficiency promoted macrophage M2 polarization by enhancing PPARγ expression.
Macrophages are abundant and critical inflammatory cells in the respiratory tract and are an essential ingredient of the mononuclear phagocyte system [8].In humans, mononuclear phagocytes are the main components of the interface between the lung and the external environment and play a key role in airway inflammation in asthma [23,24].Innate immune cells and lung epithelial cells are stimulated by allergens to create a range of cytokines that cause a disruption in the differentiation of resident lung macrophages, thereby exacerbating asthma [25].In asthma with predominantly TH2-type inflammation, the dominant macrophages are M2 macrophages, which exhibit enhanced phagocytic activity but also produce proallergic factors such as YM1 and FIZZ1 [26][27][28][29][30]. M2 macrophages can activate TH2 cells, promote eosinophil infiltration into the lung, and contribute to airway inflammatory and remodeling responses.Previous studies have found that in severe asthma, especially in patients resistant to glucocorticoid therapy, macrophages are predominantly of the M1 phenotype [31].It has been demonstrated that exosome membranes coated with EM-PLGA@Dnmt3aos smart silencer biomaterials inhibit M0 to M2 polarization by targeting M2 macrophages for the treatment of asthma [32].Furthermore, the addition of short-chain fatty acids butyrate and propionate to the diet relieves asthma by suppressing the polarization of M2 in the airways of mice [33].Preliminary evidence suggests that targeted therapy against M2 macrophages may be part of the asthma treatment.Interestingly, our results suggested that M2 macrophage polarization was promoted in MNKO mice, suggesting that macrophage NCOR1 deficiency might exacerbate asthma by enhancing M0 to M2 polarization.
The next question to be addressed is to find the pathway by which NCOR1 in macrophages regulates M2 programming and thus exacerbates asthma.PPARγ, which has been extensively studied in the field of lipid metabolism and insulin signaling, also plays a crucial role in allergic diseases such as asthma [34].In macrophages, STAT6/PPARγ signaling regulates the transcription of numerous genes, driving the M2 phenotype [35].The significant increase in M2 macrophages in the airways of asthmatic patients compared to healthy controls also suggests a potential role for PPARγ in asthma and in allergic inflammation [25].
Previous studies have reported that NCOR1 is considered a tumor suppressor gene in lung adenocarcinoma, and its mutation is associated with poor prognosis of cancer, while high expression of NCOR1 is linked to better prognosis [36].However, NCOR1 has not been studied in allergic diseases such as asthma.The role of NCOR1 in macrophages in atherosclerotic cardiovascular disease is complex and has been shown to have a protective function during atherosclerosis, but an increase in inflammatory cytokines is also observed in response to PPARγ agonist stimulation [15].Furthermore, NCOR1 deficiency was found to inhibit proinflammatory factors released in the classical activation pathway of macrophages and to enhance the expression of M2-type antiinflammatory genes in the context of myocardial infarction [20].Consistently, the study showed that macrophage NCOR1 deficiency significantly enhanced the activity of PPARγ and the expression of proinflammatory cytokines in MNKO asthmatic mice.The results of the current study identified that NCOR1 depletion in macrophages disrupted the pulmonary immune system during the development of asthma.
However, our study has limitations.First, we conducted animal experiments with mice, which have a different pathological process from humans.Second, we established a mouse asthma model with ovalbumin, whose disease characteristics were consistent with hypereosinophilic asthma in the clinic.However, non-eosinophilic asthma also exists, and we have not studied in our experiments.
In summary, NCOR1 suppresses the inflammatory response during asthma development by regulating macrophage polarization.These results convincingly highlighted the pivotal biological functions of NCOR1 in the maintenance of lung homeostasis and the pathogenesis of asthma.Thus, it provided a potential novel strategy for designing clinical therapeutic strategies against asthma.

METHODS Animals
Ncor1 flox/flox mice on a C57BL/6J background were backcrossed to mice that have lysozyme M promoter-controlled Cre recombinase expression to create Ncor1 flox/flox ;LysM cre (MNKO) mice and Ncor1 flox/flox (LC) mice [17].Genotypes of all animals were confirmed.Eight-week-old male mice were utilized in the study.All experimental operations were authorized by the Institutional Review and Ethics Board of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine.

Allergen-induced mouse asthma model
Asthma was induced in LC and MNKO mice as previously described [37,38].Briefly, on days 0, 7, and 14, mice received intraperitoneal injections of 100 g ovalbumin.Control mice received the same dosage of regular saline injection.Then, the sensitized mice were challenged on day 21 to day 28 with 50 μl of OVA (20 μg/μl) or saline under isoflurane anesthesia (Yipin, Hebei, China).

BALF
BALF was gathered by intubating and washing the lungs with 0.8 ml of cold PBS.Afterwards, samples were centrifuged at 400 × g for 5 min.After being resuspended in 1 ml of PBS, the precipitated cells were enumerated.Finally, inflammatory cells were counted by Wright-Giemsa (Baso, Zhuhai, China) staining.

Fig. 5
Fig. 5 PPARγ signaling mediates the effects of NCOR1 deficiency on M2 polarization.A blotting analysis of PPARγ in BMDMs after IL-4 and IL-13 stimulation.B Quantification of western blotting results exemplified in (A).C Western blotting analysis of Arginase 1 and PPARγ in BMDMs treated with IL-4 and IL-13 with the PPARγ inhibitor T0070907.D, E Quantification of western blotting results exemplified in (C).F Western blotting analysis of Arginase 1 and PPARγ in BMDMs treated with IL-4 and IL-13 with Scr siRNA or Pparγ siRNA.G, H Quantification of western blotting results exemplified in (F).I, J RT-QPCR analysis of PPARγ and Arginase 1 in BMDMs treated with IL-4 and IL-13 with Scr siRNA or Pparγ siRNA.n = 3:3:3:3.n.s not significant.*p < 0.05, **p < 0.01, ***p < 0.001.