Cereblon Deficiency Contributes to the Development of Elastase-Induced Emphysema by Enhancing NF-κB Activation

Cereblon (CRBN) has been shown to play an essential role in regulating inflammatory response and endoplasmic reticulum stress, thus mediating the development of various diseases. However, little is known about the roles of CRBN in chronic obstructive pulmonary disease (COPD) pathogenesis. We found that the protein levels of CRBN in lung homogenates from patients with COPD were lower than those from never smokers and smokers. The CRBN protein level was positively correlated with the forced expiratory volume in 1 s (FEV1)/forced vital capacity (FVC). To investigate the role of CRBN in modulating elastase-induced emphysema, we used Crbn knockout (KO) mice. Elastase-induced emphysematous changes were significantly aggravated in Crbn KO mice. Neutrophil infiltration, lung cell injury, and protein leakage into the bronchoalveolar space were more severe in Crbn KO mice than in wild-type (WT) mice. Furthermore, Crbn KO resulted in the elevated release of neutrophilic chemokines and inflammatory cytokines in lung epithelial cells and macrophages. The transcriptional activity of nuclear factor-κB (NF-κB) was significantly increased in Crbn knocked-down cells. In conclusion, Crbn deficiency might be involved in the development of emphysema by enhancing NF-κB activation, suggesting that targeting CRBN might be an effective therapeutic approach for the treatment of COPD.


Introduction
Chronic obstructive pulmonary disease (COPD) is a major healthcare problem worldwide. The Global Burden of Disease Study 2015 estimated the global prevalence of COPD at approximately 174 million cases, and COPD ranked third among fatal diseases with approximately 3.2 million patients dying of the disease [1]. Emphysema is a primary phenotype of COPD and is defined as the irreversible destruction of alveolar structures and enlargement of the airspaces [2]. Oxidant/antioxidant or protease/antiprotease imbalances are pivotal to the pathogenesis of emphysema [3]. Considerable efforts have been made over the past decade to find a novel therapeutic to slow the inflammation of the lung and the progression of emphysema [4]. However, there is no effective treatment to change the natural course of emphysema because of the complexity of inflammatory signaling and mechanisms [5].
Cereblon (CRBN) was initially discovered as a candidate gene for a mild form of autosomal recessive non-syndromic mental retardation, and it has since been reported as a direct molecular target for the teratogenicity of thalidomide and the cytotoxicity of immunomodulatory drugs [6,7]. CRBN has been extensively studied and found to regulate

Analysis of Bronchoalveolar Lavage Fluid (BALF)
On days 2 and 14 after elastase administration, the lungs of terminally anesthetized mice were lavaged with 1 mL of cold phosphate-buffered saline (PBS). BALF was centrifuged at 1500 rpm at 4 • C for 10 min and the supernatants were then collected for the measurement of the levels of lactate dehydrogenase (LDH) and protein. The total cell count was determined with a hemocytometer and BAL cell distribution was quantified in cytospin preparations after staining with Diff-Quik dye (Sysmex, Kobe, Japan).

Emphysema Measurement and Immunohistochemistry
The right lungs were fixed in 4% paraformaldehyde solution (USB products, USA) for 24 h, embedded in paraffin, and sectioned in 4 µm-thick slices. The slides were stained with hematoxylin and eosin (H&E). Four randomly selected ×100 fields per specimen were photographed in a blinded manner. Emphysema was quantified by measuring the mean linear intercept (MLI). The MLI was measured by placing four 1000-µm lines over each field. The total length of each line divided by the number of alveolar intercepts gives the average distance. The non-parenchymal area was not included. Lung tissues were placed on slides using the Discovery XT automated immunohistochemistry stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA). Tissue sections were deparaffinized and rehydrated. Cell conditioning 1 standard (pH 8.4 buffer containing Tris/borate/EDTA) was used for antigen retrieval. The sections were incubated with anti-8-hydroxy-2 -deoxyguanosine (8-OHdG), anti-4 hydroxynonenal (4HNE), or anti-matrix metallopeptidase 9 (MMP9) antibody for 32 min at 37 • C, washed, and incubated with a secondary antibody for 20 min at 37 • C.

Preparation of Cigarette Smoke Extract (CSE)
Commercial cigarettes (THIS; KT&G Corp., Daejeon, South Korea) were smoked continuously by a bottle system connected to a vacuum machine (Gast Manufacturing Inc., Benton Harbor, MI, USA). The smoke from 20 cigarettes was bubbled in 60 mL of PBS. The large insoluble particles were removed by filtering the solution through a 0.22 µm filter.

Protein Extraction and Western Blot Analysis
Total cellular extracts were prepared using 1× Cell Lysis Buffer (Cell Signaling Technology) supplemented with 1 mM PMSF. Frozen lung tissues (SNUH IRB Number: H-1309-073-521) were homogenized in tissue extraction buffer (Life Technologies) contain-Antioxidants 2022, 11,1980 4 of 17 ing a protease inhibitor mixture (Sigma) and phosphatase inhibitor mixture (Sigma). Protein concentration was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). Cell extracts were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and gels were transferred to Hybond ECL nitrocellulose membranes (Thermo Fisher Scientific) for 100 min at 90 V. The membranes were blocked with 5% skim milk in 1× Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBS-T) for 1 h at room temperature. After successful washes, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Blots were developed using a West Pico Western blot detection kit (Thermo Fisher Scientific).

Luciferase Assay
Cells were transfected with the NF-κB reporter plasmid or control plasmid using a Neon Transfection System (Thermo Fisher Scientific) according to the manufacturer's specifications. Luciferase activity was determined using a luciferase assay kit (Promega, Madison, WI, USA).

Statistical Analysis
Data were analyzed using Prism (version 5; GraphPad Software, San Diego, CA, USA). Differences between means were explored using the Kruskal-Wallis test via the Dunn's multiple comparison post hoc test. To evaluate correlations in the human samples, the Pearson correlation coefficient was calculated. Differences between groups were evaluated by Student's t-test. A p-value of <0.05 was considered significant.

The Expression Level of CRBN Was Decreased in Lung Tissues of Patients with COPD and Correlated with the Forced Expiratory Volume in 1 s (FEV1)/Forced Vital Capacity (FVC) Ratio
To investigate the functional role of CRBN in emphysema development in the human lung, we first measured CRBN expression in surgically resected human lung tissues with immunoblotting. The expression of CRBN protein was lower in COPD lungs (n = 8) than in non-COPD lungs (never smokers; n = 8, smokers; n = 8) (Figure 1a,b). CRBN expression was not significantly different between never smokers and smokers (Figure 1a,b). Cigarette smoking is the main risk factor for COPD, yet only 20-30% of smokers develop COPD [18]. These findings suggest that CRBN plays a role in the development of COPD regardless of smoking. We further investigated the association between clinical parameters and the level of CRBN expression. The clinical parameters included age, body mass index, pack-years of smoking (py), pre-bronchodilator FEV1, the ratio of FEV1 to FVC, diffusion capacity (DLco), COPD assessment test (CAT) score, St. George's Respiratory Questionnaire (SGRQ), and Modified Medical Research Council scale (mMRC). Among these clinical parameters, only FEV1/FVC was significantly correlated with the level of CRBN expression (Figure 1c-k). A FEV1/FVC ratio of less than 70% indicated airflow limitation and the possibility of COPD. The correlation between the ratio of FEV1/FVC and the expression of CRBN showed that a decreased level of CRBN is consistently associated with COPD development.

Crbn KO Exaggerated Elastase-Induced Emphysema in Mice
Rodent models of elastase-induced emphysema were established [19]. To evaluate whether the expression of CRBN affected the development of elastase-induced emphysema in mice, we treated WT and Crbn KO BALB/c mice with elastase intratracheally. Crbn KO was confirmed via RT-PCR analysis (Figure 2a). On day 14 after intratracheal elastase administration, more prominent alveolar destruction and airspace enlargement were observed in elastase-treated mice than in saline-treated mice. These changes were more exaggerated in Crbn KO mice than in WT mice (Figure 2b). Consistent with the morphological results, the MLI (a measurement of the mean interalveolar septal wall distance) was markedly higher in the lungs of elastase-treated Crbn KO mice than in the lungs of WT mice (Figure 2c).

Crbn KO Exaggerated Elastase-Induced Emphysema in Mice
Rodent models of elastase-induced emphysema were established [19]. To evaluate whether the expression of CRBN affected the development of elastase-induced emphysema in mice, we treated WT and Crbn KO BALB/c mice with elastase intratracheally. Crbn KO was confirmed via RT-PCR analysis (Figure 2a). On day 14 after intratracheal elastase administration, more prominent alveolar destruction and airspace enlargement were observed in elastase-treated mice than in saline-treated mice. These changes were more exaggerated in Crbn KO mice than in WT mice ( Figure 2b). Consistent with the morphological results, the MLI (a measurement of the mean interalveolar septal wall distance) was  Representative images of H&E staining in lungs from WT and Crbn KO mice 14 days after intratracheal instillation of elastase (×40). (c) The MLI was measured. Data represent means ± SE. * p < 0.05, and *** p < 0.001.

Crbn KO Increased Elastase-Induced Inflammation and Cellular Injury
The lungs of elastase-treated mice showed marked inflammation with immune cell infiltration on day 2 that returned to basal levels on day 14. Elastase-induced inflammation and emphysema were more prominent in Crbn KO mice than in WT mice ( Figure 3a). To investigate whether elastase-induced inflammation mimicked those of patients with COPD, we analyzed bronchoalveolar lavage fluid (BALF). Neutrophils are the most abundant inflammatory cells in the bronchi of patients with COPD, and increased neutrophilic inflammation is characteristic of acute exacerbation of COPD [20]. The total number of BALF cells was significantly higher on day 2 after elastase administration in Crbn KO mice than in WT mice ( Figure 3b). Along with the total cell count, neutrophils were markedly increased in BALF from elastase-treated Crbn KO mice ( Figure 3c). However, most of the inflammatory cells in the BALF disappeared 14 days after elastase administration, when

Crbn KO Increased Elastase-Induced Inflammation and Cellular Injury
The lungs of elastase-treated mice showed marked inflammation with immune cell infiltration on day 2 that returned to basal levels on day 14. Elastase-induced inflammation and emphysema were more prominent in Crbn KO mice than in WT mice ( Figure 3a). To investigate whether elastase-induced inflammation mimicked those of patients with COPD, we analyzed bronchoalveolar lavage fluid (BALF). Neutrophils are the most abundant inflammatory cells in the bronchi of patients with COPD, and increased neutrophilic inflammation is characteristic of acute exacerbation of COPD [20]. The total number of BALF cells was significantly higher on day 2 after elastase administration in Crbn KO mice than in WT mice (Figure 3b). Along with the total cell count, neutrophils were markedly increased in BALF from elastase-treated Crbn KO mice (Figure 3c). However, most of the inflammatory cells in the BALF disappeared 14 days after elastase administration, when the emphysematous changes were established. The concentration of LDH ( Figure 3d) and protein (Figure 3e) in the BALF were significantly higher in elastase-treated Crbn KO mice than in elastase-treated WT mice. On day 14 after elastase administration, the levels of LDH Antioxidants 2022, 11, 1980 7 of 17 and protein in BALF were reduced to the baseline in the same manner as the neutrophils. We measured the levels of neutrophilic chemokines and inflammatory cytokines: KC, TNFα, IL-6, and IL-1β in total lung tissues (Figure 3f-i) and BALF (3j-m). The expression levels of KC and TNF-α mRNA were significantly higher on day 2 after elastase administration in Crbn KO mice than in WT mice (Figure 3f,g). Only IL-6 in BALF was higher in elastasetreated Crbn KO mice than in elastase-treated WT mice (Figure 3l). These findings suggested that neutrophilic inflammation and cellular injury precede emphysema development, and CRBN deficiency could contribute to elastase-induced neutrophilic inflammation, leading to cellular injury.

Crbn KO Enhanced Production of Inflammatory Cytokines and Chemokines by Increasing NF-κB Activation in Lung Epithelial Cells and Macrophages
Interleukin-8 (IL-8) is a neutrophil chemoattractant whose levels in sputum samples of patients with COPD are increased [21]. To investigate the role of CRBN on IL-8 production, we transiently transfected BEAS-2B cells with the control and Crbn siRNAs and treated the cells with NE (1 U/mL) or CSE (1%) for 24 h. NE or CSE treatment induced IL-8 production in BEAS-2B cells. NE or CSE-induced IL-8 production was higher in Crbn siRNA-transfected cells than in the control siRNA-transfected cells (Figure 4a,b).
Antioxidants 2022, 11, x FOR PEER REVIEW 7 of 17 the emphysematous changes were established. The concentration of LDH ( Figure 3d) and protein (Figure 3e) in the BALF were significantly higher in elastase-treated Crbn KO mice than in elastase-treated WT mice. On day 14 after elastase administration, the levels of LDH and protein in BALF were reduced to the baseline in the same manner as the neutrophils. We measured the levels of neutrophilic chemokines and inflammatory cytokines: KC, TNF-α, IL-6, and IL-1β in total lung tissues (Figure 3f-i) and BALF (3j-m). The expression levels of KC and TNF-α mRNA were significantly higher on day 2 after elastase administration in Crbn KO mice than in WT mice (Figure 3f,g). Only IL-6 in BALF was higher in elastase-treated Crbn KO mice than in elastase-treated WT mice (Figure 3l). These findings suggested that neutrophilic inflammation and cellular injury precede emphysema development, and CRBN deficiency could contribute to elastase-induced neutrophilic inflammation, leading to cellular injury.

Crbn KO Enhanced Production of Inflammatory Cytokines and Chemokines by Increasing NF-κB Activation in Lung Epithelial Cells and Macrophages
Interleukin-8 (IL-8) is a neutrophil chemoattractant whose levels in sputum samples of patients with COPD are increased [21]. To investigate the role of CRBN on IL-8 production, we transiently transfected BEAS-2B cells with the control and Crbn siRNAs and treated the cells with NE (1 U/mL) or CSE (1%) for 24 h. NE or CSE treatment induced IL-8 production in BEAS-2B cells. NE or CSE-induced IL-8 production was higher in Crbn siRNA-transfected cells than in the control siRNA-transfected cells (Figure 4a,b).
Because alveolar macrophages play a central role in modulating inflammation by producing many inflammatory proteins in patients with COPD [22], we next investigated the effect of Crbn KO not only in bronchial epithelial cells, but also in macrophages. BMDMs from WT and Crbn KO mice were treated with NE (1 U/mL), CSE (1%), or LPS (100 ng/mL). We measured the concentrations of KC, MIP2, TNF-α, and IL-6 in the supernatant of BMDMs. In NE-treated BMDMs, the concentrations of KC, MIP2, TNF-α, and IL-6 were not significantly different between WT and Crbn KO mice (Figure 4c-f). In CSEtreated BMDMs, KC and MIP2 were significantly higher in Crbn KO cells than in WT cells (Figure 4g,h). However, CSE induced neither TNF-α nor IL-6 (Figure 4i,j). In LPS-treated BMDMs, the production of KC, MIP2, TNF-α, and IL-6 was significantly higher in Crbn KO cells than in WT cells (Figure 4k-n).
Many inflammatory cytokines/chemokines are regulated by the mitogen-activated protein kinases (MAPKs) and NF-κB pathways [23]. Given the increased neutrophilic inflammation in Crbn KO mice and the increased release of inflammatory cytokines/chemokines in Crbn-deficient cells, we investigated the effect of decreased levels of CRBN on the activation of MAPKs and NF-κB. The activation of the MAPK pathway was Because alveolar macrophages play a central role in modulating inflammation by producing many inflammatory proteins in patients with COPD [22], we next investigated the effect of Crbn KO not only in bronchial epithelial cells, but also in macrophages. BMDMs from WT and Crbn KO mice were treated with NE (1 U/mL), CSE (1%), or LPS (100 ng/mL). We measured the concentrations of KC, MIP2, TNF-α, and IL-6 in the supernatant of BMDMs. In NE-treated BMDMs, the concentrations of KC, MIP2, TNF-α, and IL-6 were not significantly different between WT and Crbn KO mice (Figure 4c-f). In CSE-treated BMDMs, KC and MIP2 were significantly higher in Crbn KO cells than in WT cells (Figure 4g,h). However, CSE induced neither TNF-α nor IL-6 (Figure 4i,j). In LPS-treated BMDMs, the production of KC, MIP2, TNF-α, and IL-6 was significantly higher in Crbn KO cells than in WT cells (Figure 4k-n).
Many inflammatory cytokines/chemokines are regulated by the mitogen-activated protein kinases (MAPKs) and NF-κB pathways [23]. Given the increased neutrophilic inflammation in Crbn KO mice and the increased release of inflammatory cytokines/chemokines in Crbn-deficient cells, we investigated the effect of decreased levels of CRBN on the activation of MAPKs and NF-κB. The activation of the MAPK pathway was determined with the Western blot analysis of phosphorylated MAPKs (ERK, JNK, and p38). The NF-κB activation process involves IκBα phosphorylation by IκB kinases (IKKs), and the subsequent degradation of IκBα, and nuclear translocation of NF-κB [23]. Therefore, activation of the NF-κB pathway was determined with a Western blot analysis of IκBα and luciferase activity assay in cells transfected with an NF-κB-luciferase reporter construct. Both NE and CSE induced IκBα degradation and MAPK activation, which were not affected by Crbn deficiency (Figure 5a,b, Supplementary Figure S1a-d). LPS-induced degradation of IκBα also was not changed by Crbn KO (Figure 5c). However, NF-κB transcriptional activity was significantly increased in Crbn knocked-down cells treated with CSE ( Figure 5d). These results suggest that exaggerated inflammation in Crbn KO mice and cells might be due to increased activation of the NF-κB pathway, probably following IκBα degradation.
fore, activation of the NF-κB pathway was determined with a Western blot analysis of IκBα and luciferase activity assay in cells transfected with an NF-κB-luciferase reporter construct. Both NE and CSE induced IκBα degradation and MAPK activation, which were not affected by Crbn deficiency (Figure 5a,b, Supplementary Figure 1a-d). LPS-induced degradation of IκBα also was not changed by Crbn KO (Figure 5c). However, NF-κB transcriptional activity was significantly increased in Crbn knocked-down cells treated with CSE ( Figure 5d). These results suggest that exaggerated inflammation in Crbn KO mice and cells might be due to increased activation of the NF-κB pathway, probably following IκBα degradation.  with CSE (1%) for 24 h. (k-n) WT and Crbn KO BMDMs were treated with LPS (100 ng/mL) for 6 or 24 h. The levels of inflammatory cytokines/chemokines in cell culture media were measured with an ELISA. Data represent the mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Crbn KO Increased Elastase-Induced Oxidative Damage and MMP9 Expression
Oxidative stress is recognized as an important predisposing factor in COPD pathogenesis, [24] and MMPs are known to contribute to elastin and collagen matrix degradation, resulting in emphysema [25]. To elucidate whether Crbn KO affects elastase-induced oxidative damage, we performed immunohistochemical staining for 8-OHdG (one of the major products of DNA oxidation) and 4HNE (a product of lipid peroxidation) in lung tissues from elastase-instilled WT and Crbn KO mice. The intensity of 8-OHdG and 4HNE staining on day 2 after elastase instillation was greater in Crbn KO mice than in WT mice (Figure 6a-d). MMP-9 is one of the predominant elastolytic enzymes in patients with COPD [26]. Increased intensity of MMP9 staining was found in the lungs of elastasetreated Crbn KO mice, and the MMP9-positive cells were mostly recruited immune cells (Figure 7a,b). Furthermore, NE-or LPS-induced MMP9 expression was increased in Crbn-deficient lung epithelial cells and macrophages (Figure 7c,d). These results indicate that Crbn deficiency could also promote elastase-induced oxidative stress and proteinase expression.

Crbn KO Increased Elastase-Induced Oxidative Damage and MMP9 Expression
Oxidative stress is recognized as an important predisposing factor in COPD pa genesis, [24] and MMPs are known to contribute to elastin and collagen matrix degr tion, resulting in emphysema [25]. To elucidate whether Crbn KO affects elastase-indu oxidative damage, we performed immunohistochemical staining for 8-OHdG (one o major products of DNA oxidation) and 4HNE (a product of lipid peroxidation) in l tissues from elastase-instilled WT and Crbn KO mice. The intensity of 8-OHdG and 4H staining on day 2 after elastase instillation was greater in Crbn KO mice than in WT m (Figure 6a-d). MMP-9 is one of the predominant elastolytic enzymes in patients w COPD [26]. Increased intensity of MMP9 staining was found in the lungs of elast treated Crbn KO mice, and the MMP9-positive cells were mostly recruited immune (Figure 7a,b). Furthermore, NE-or LPS-induced MMP9 expression was increased in C deficient lung epithelial cells and macrophages (Figure 7c,d). These results indicate Crbn deficiency could also promote elastase-induced oxidative stress and proteinase pression.

Discussion
The role of CRBN in the pathogenesis of COPD has not been investigated. Our present findings show that decreased CRBN contributes to the development of emphysema by increasing neutrophilic inflammation, oxidative damage, and proteinase (MMP9) expression. One plausible mechanism of increased inflammation is that CRBN enhances the transcriptional activity of NF-κB in lung epithelial cells and macrophages.
Neutrophilic inflammation is a prominent feature in the COPD airway, and neutrophil products are thought to be key mediators causing emphysema [27]. Airway neutrophilia is associated with the rate of decline in lung function [28]. This is supported by the finding that the protein expression of CRBN in total lung tissue lysates from patients with COPD was lower than that in lung tissue lysates of both never smokers and smokers. The FEV1/FVC ratio, which is an essential parameter for COPD diagnosis, was significantly correlated with CRBN levels. Lung function parameter, especially FEV1/FVC, correlates with the extent of emphysema [29]. This suggests COPD patients with lower CRBN levels

Discussion
The role of CRBN in the pathogenesis of COPD has not been investigated. Our present findings show that decreased CRBN contributes to the development of emphysema by increasing neutrophilic inflammation, oxidative damage, and proteinase (MMP9) expression. One plausible mechanism of increased inflammation is that CRBN enhances the transcriptional activity of NF-κB in lung epithelial cells and macrophages.
Neutrophilic inflammation is a prominent feature in the COPD airway, and neutrophil products are thought to be key mediators causing emphysema [27]. Airway neutrophilia is associated with the rate of decline in lung function [28]. This is supported by the finding that the protein expression of CRBN in total lung tissue lysates from patients with COPD was lower than that in lung tissue lysates of both never smokers and smokers. The FEV1/FVC ratio, which is an essential parameter for COPD diagnosis, was significantly correlated with CRBN levels. Lung function parameter, especially FEV1/FVC, correlates with the extent of emphysema [29]. This suggests COPD patients with lower CRBN levels have more emphysematous lung. As expected, in the elastase-treated mouse model, an exaggerated emphysematous change was observed in Crbn KO mice compared to WT mice. More neutrophils were recruited to the lungs, and lung cell damage as indicated by LDH release and protein leakage into BALF was more severe in elastase-treated Crbn KO mice than in elastase-treated WT mice. Resident lung cells, such as epithelial cells and alveolar macrophages, play an essential role in initiating and modulating inflammation by producing a variety of inflammatory cytokines and chemokines in patients with COPD [22]. Therefore, we investigated the role of CRBN in inflammatory responses in both cell types.
Because cigarette smoking is the major risk factor for COPD, we stimulated both cell types with CSE as well as with elastase. Elastase or CSE-induced IL-8 production was significantly enhanced with Crbn knockdown in lung epithelial cells. Although elastase alone did not induce KC, MIP2, TNF-α, or IL-6 in WT or Crbn KO BMDMs, CSE-mediated secretion of neutrophilic chemokines such as KC and MIP2 was exaggerated in Crbn KO BMDMs. NE was reported to upregulate inflammatory cytokine production in macrophages differentiated from primary peripheral blood mononuclear cells [30]. Similar to our results, NE treatment alone did not increase the levels of TNF-α and IL-1β. However, in response to LPS treatment, NE enhanced the release of those cytokines [30]. In chronic inflammatory environments such as COPD, multiple factors interact to stimulate inflammatory responses. NE alone does not seem enough to induce an inflammatory response. Cigarette smoke (CS) and CS extracts (CSE) were known to induce inflammatory cytokines in several types of cells including lung epithelial cells and macrophages. However, in this study, CSE did not induce the release of proinflammatory cytokines, such as TNF-α and IL-6 in BMDM. In our previous study, we performed a series of experiments to explain the discrepancy in cytokine production by CSE [31]. As well as in BMDM, incubation with CSE did not increase the release of TNF-α, IL-6, and IL-1β in RAW264.7 cells, peritoneal macrophages, and bronchoalveolar lavage (BAL) macrophages. Co-treatment with CSE significantly suppressed the levels of TNF-α, IL-6, and IL-1β mRNA/protein in LPS-treated RAW264.7 cells, peritoneal macrophages, and BAL macrophages via p300 downregulation [31]. We do not know exactly why CSE did not increase inflammatory cytokine production unlike the findings of other researchers, but a non-standardized CSE extraction method or the use of other types of cigarettes might be the cause. Therefore, to clarify the role of CRBN on the production of inflammatory chemokines and cytokines in macrophage, we used LPS as a stimulus.
LPS is a toxic component of Gram-negative bacteria that provokes profound inflammation [32]. Patients with COPD with exacerbations have a significantly increased number of inflammatory cells in the sputum and an accelerating progression of emphysema compared with patients with COPD without exacerbations [33,34]. Most exacerbations are precipitated by viral or bacterial infections, and Gram-negative bacteria are the most commonly isolated pathogens in such cases [35]. Even a single intratracheal administration of LPS to mice with elastase-induced emphysema can provoke inflammation, increase protease production, and finally lead to more severe emphysematous change [36]. We also evaluated the effect of CRBN in LPS-treated WT and Crbn KO BMDMs. LPS strongly induced inflammatory chemokines/cytokines, which were remarkably enhanced in Crbn KO BMDMs. This finding suggests that CRBN may have a role in acute inflammatory response as well as chronic inflammation.
CSE-induced IL-8 production, and LPS-induced proinflammatory cytokines are known to be regulated by the MAPK and NF-κB pathways [37][38][39]. However, MAPK activation by elastase or CSE was not affected by CRBN expression. NF-κB activation involves IκBα phosphorylation by IKKs and the subsequent degradation of IκBα. NF-κB liberated from the IκB complex translocates into the nucleus, resulting in the expression of many inflammatory genes, including those encoding TNF-α and interleukins [37,39]. Although Crbn KO did not influence IκBα degradation in cells treated with elastase, CSE, or LPS in our study, the transcriptional activity of NF-κB was enhanced when the level of CRBN was reduced. In this regard, Crbn KO might increase neutrophilic inflammation and lead to profound emphysema by enhancing NF-κB activation.
In addition to inflammation, oxidative stress, proteinase/anti-proteinase imbalance, activation of autophagy, cellular senescence, and apoptosis are also considered important pathogenic factors of COPD [40]. MMPs and the lipid peroxidation product 4HNE are reported to be elevated in lung tissues of patients with COPD [41]. Consistent with this, the lungs of elastase-treated mice contained elevated numbers of cells positive for 8-OHdG, 4HNE, and MMP9, an effect that was augmented with KO of Crbn. Because oxidative stress, proteinase activity, and inflammation are reciprocally regulated, CRBN appears to regulate each process either directly or indirectly. However, a reduced level of CRBN did not affect CSE-induced cellular aging, activation of autophagy, or apoptosis in lung epithelial cells (Supplementary Figure S2a-c). Class A scavenger receptors (SR-A) are mainly expressed in macrophages. SR-A could increase inflammation, [42] and COPD is associated with SR-A gene sequence variation [43]. However, Crbn KO did not change the levels of SR-As as in SR-A1 or the macrophage receptor with the collagenous structure (MARCO) in LPS-or elastase-treated BMDMs (Supplementary Figures S3 and S4).

Conclusions
In conclusion, Crbn deficiency contributes to the development of emphysema by increasing neutrophilic inflammation, oxidative damage, and proteinase expression (Figure 8). This study provides new evidence for the therapeutic potential of targeting CRBN for the treatment of COPD/emphysema. However, the molecular mechanism of how CRBN regulates the transcriptional activity of NF-κB in lung epithelial cells and macrophages is unclear. To elucidate the mechanism, further detailed studies are required. Moreover, if the role of CRBN in acute inflammatory conditions is investigated, it would be helpful to regulate COPD exacerbation. CRBN was reduced. In this regard, Crbn KO might increase neutrophilic inflammation and lead to profound emphysema by enhancing NF-κB activation. In addition to inflammation, oxidative stress, proteinase/anti-proteinase imbalance, activation of autophagy, cellular senescence, and apoptosis are also considered important pathogenic factors of COPD [40]. MMPs and the lipid peroxidation product 4HNE are reported to be elevated in lung tissues of patients with COPD [41]. Consistent with this, the lungs of elastase-treated mice contained elevated numbers of cells positive for 8-OHdG, 4HNE, and MMP9, an effect that was augmented with KO of Crbn. Because oxidative stress, proteinase activity, and inflammation are reciprocally regulated, CRBN appears to regulate each process either directly or indirectly. However, a reduced level of CRBN did not affect CSE-induced cellular aging, activation of autophagy, or apoptosis in lung epithelial cells (Supplementary Figure S2a-c). Class A scavenger receptors (SR-A) are mainly expressed in macrophages. SR-A could increase inflammation, [42] and COPD is associated with SR-A gene sequence variation [43]. However, Crbn KO did not change the levels of SR-As as in SR-A1 or the macrophage receptor with the collagenous structure (MARCO) in LPS-or elastase-treated BMDMs (Supplementary Figures S3 and S4).

Conclusions
In conclusion, Crbn deficiency contributes to the development of emphysema by increasing neutrophilic inflammation, oxidative damage, and proteinase expression ( Figure  8). This study provides new evidence for the therapeutic potential of targeting CRBN for the treatment of COPD/emphysema. However, the molecular mechanism of how CRBN regulates the transcriptional activity of NF-κB in lung epithelial cells and macrophages is unclear. To elucidate the mechanism, further detailed studies are required. Moreover, if the role of CRBN in acute inflammatory conditions is investigated, it would be helpful to regulate COPD exacerbation. Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Crbn Knockdown does not affect NE-and CSE-induced degradation of IκBα.; Figure S2: Crbn Knockdown does not affect CSE-induced aging, activation of autophagy, and apoptosis; Figure S3: Crbn KO did not affect the LPS-induced surface expression levels of scavenger receptors in BMDM; Figure S4: Crbn KO did not affect the LPS-induced surface expression levels of scavenger receptors in elastase treated BMDM.