Involvement of the Constitutive Photomorphogenesis 9 Signalosome Subunit 5 With Programmed Cell Death Protein 1 Ligand in Asthma

Abstract

Purpose

The constitutive photomorphogenesis 9 signalosome (CSN) is a highly conserved protein complex comprised of eight subunits, each of which play crucial roles in diverse cellular processes, such as signal transduction, gene transcription, angiogenesis, and cell proliferation. In the context of asthma, a potential emerging target is the programmed death-ligand 1 (PD-L1)-mediated pathway, which serves as a significant immune checkpoint inhibitor in this condition. However, the precise involvement of CSN subunit 5 (CSN5) in bronchial asthma and the interplay between CSN5 and PD-L1 in asthma remain poorly understood.

Methods

The potential association between CSN5 and bronchial asthma was explored in a mouse model of ovalbumin (OVA)-induced asthma. Samples were obtained from human lung microvascular endothelial cell (HMVEC-L) treated with Dermatophagoides pteronyssinus (Der p 1) and CSN5 small interfering RNA. The expression of nuclear factor (NF)-κB, IκBα, inhibitor of κB kinase β (IKKβ), PD-L1, and CSN5 was assessed. Additionally, plasma CSN5 levels in asthma patients, both in stable and exacerbated states, were examined.

Results

Plasma levels of CSN5 were elevated in patients with exacerbated asthma (n = 19) compared to both healthy controls (n = 10) and patients with stable asthma (n = 19). The CSN5 level demonstrated a correlation with lung function in individuals with asthma. Silencing CSN5 in HMVEC-L led to a reduction in NF-κB protein levels at 4 hours and PD-L1 levels at 4, 8, and 24 hours after Der p 1 treatment. In OVA-sensitized/challenged mice, goblet cell hyperplasia, lung fibrosis, and the levels of CSN5, PD-L1, interleukin-13, interferon-γ, phospho (p)-NF-κB, p-IκBα, and p-IKKβ proteins increased at 33 and 80 days compared to control mice. However, these changes were mitigated by treatment with a PD-L1 inhibitor.

Conclusions

These findings suggest that CSN5, along with PD-L1, could serve as a promising target for the treatment of asthma.

INTRODUCTION

Asthma is currently recognized as a complex condition with varying severity, natural history, and response to treatment. Asthma has traditionally been described as a condition, marked by inflammation of the airways involving eosinophils, and associated with increased sensitivity of the bronchi. The long past T helper (Th)1/Th2 paradigm has been pivotal in understanding asthma pathogenesis, particularly in severe cases, where the control of Th1/Th2 cytokine formation differs compared to mild and moderate asthma.¹,²,³ The genes associated with virus detection, bacterial infection, restructuring of lung tissue, and inflammatory processes involving eosinophils and neutrophils, as well as the genes within the asthma susceptibility locus on chromosome 17q12, were identified in transcriptome-wide asthma association studies.⁴

Jab1, also known as constitutive photomorphogenesis 9 signalosome subunit 5 (CSN5), was initially identified as c-Jun-activation-domain binding protein-1, primarily facilitating AP-1-dependent gene transcription by stabilizing c-Jun.⁵,⁶ Because of this role, CSN5 is involved in various cellular processes, such as DNA repair, apoptosis, and tumorigenesis.⁷,⁸ Rising evidence indicates that CSN5 is a positive regulator for oncogenes and a negative modulator of tumor suppressors in heterogenous human malignancies.⁷,⁹ CSN5 tissue expression was highly associated with metastasis and poor prognosis in patients with pancreatic cancer and osteosarcoma.¹⁰,¹¹

CSN5 expression was higher in non-small cell lung carcinoma (NSCLC) tissues compared to the matching non-tumor tissues. Elevated CSN5 expression is strongly associated with tumor progression and unfavorable survival outcomes in NSCLC.¹² CSN5 functions as an oncogenic gene in NSCLC, suggesting its potential as a diagnostic and therapeutic target for the disease.¹³

It is reported that inflammation-influenced and nuclear factor (NF)-κB mediated expression of the deubiquitinating enzyme CSN5 leads to the stabilization of the programmed death-ligand 1 (PD-L1) and immune blocking in cancers.¹⁴,¹⁵

No studies have yet examined the role of CSN5 in asthma pathogenesis. Asthma and chronic rhinosinusitis with nasal polyps exhibit a shared inflammatory profile characterized by the involvement of Th2 lymphocytes. The activity of T-cells can be modulated through the programmed cell death protein 1 (PD-1) receptor, influencing the immune response.¹⁶

PD-L1 may contribute to the Th17/interleukin (IL)-17 immune response, which is associated with neutrophilic asthmatic inflammation. A PD-L1 blockade reduces neutrophils and mucus production in the lungs.¹⁷ PD-1 and its ligands revealed a crucial costimulatory function of PD-L1 and PD-L2 in asthma pathogenesis. PD-L1 is involved in the maintenance of peripheral tolerance, and contributes to the induction of asthma. PD-L1 and PD-L2 have opposing roles in modulating and polarizing T-cell functions in airway hyperresponsiveness (AHR) and airway inflammation in an animal model of asthma.¹⁸

There was no study of the interaction between CSN5 and PD-L1 in asthma. And the biological characters and fundamental roles of CSN5 in asthma remain largely undetermined. In this research, to clarify the role of CSN5 and PD-L1 in asthma, CSN5 expression was assessed in endothelial cells and a murine asthma model. Additionally, the relationship between CSN5 expression and clinical profiles in asthmatics patients was examined.

MATERIALS AND METHODS

Experimental design

Eight BALB/c mice were exposed to saline (normal control, NC) or ovalbumin (OVA) and treated with dexamethasone (5 mg/kg) or a PD-L1 inhibitor; nivolumab (Opdivo inj) (3 mg/kg). Detailed analyses included measurements of cytokine expression and lung CSN5 and PD-L1 protein levels. Histologic examination, hematoxylin and eosin (H&E) staining, periodic acid–Schiff (PAS) staining, Masson’s trichrome staining, immunohistochemistry, and collagen assays were performed in lung tissue. Human lung microvascular endothelial cell (HMVEC-L) with CSN5 gene silencing were treated with Dermatophagoides pteronyssinus (Der p 1).

Patients and control subjects

A total of 19 asthmatic patients with paired plasma samples were collected from the Bucheon Hospital of Soonchunhyang University, a member of the Korean Biobank Network. Asthma diagnosis and exacerbation were based on the Global Initiative for Asthma guidelines.¹⁹ Normal control subjects were recruited from the spouses of the subjects or members of the general population. The clinical characteristics of the patients and healthy individuals are presented in Table 1. This study was approved by the Institutional Review Board of Soonchunhyang University Hospital (approval No. SCHBC 2017-12-013-003).

Table 1. Clinical characteristics of the control subjects and patients with asthma.

Variables		Healthy controls	Stable asthma	Exacerbated asthma
No. of subjects		10	19
Sex
	Male	2 (20.0)	4 (21.1)
	Female	8 (80.0)	15 (78.9)
Age (at initial visit) (yr)		65.2 ± 8.7	63.2 ± 17.04
Age of asthma onset (yr)		-	57.7 ± 17.7
Asthma duration (yr)		-	5.47 ± 4.65
Smoking status
	Non-smoker	10 (100.0)	14 (73.7)
	Ex-smoker	0 (0.0)	2 (10.5)
	Smoker	0 (0.0)	3 (15.8)
Smoking amount (pack-year)		-	25 (15–40)
Lung function
	FVC % pred.	92 ± 21	88.58 ± 13.42	69.21 ± 13.16*,†
	FEV1, % pred.	112.2 ± 22.04	86.21 ± 16.08*	58.58 ± 18.45*,†
	FEV1/FVC	87.1 ± 6.74	69.44 ± 11.22*	60.00 ± 13.64*,†
BMI (kg/m2)		23.88 ± 3.12	24.45 ± 4.74
PC20 (mg/mL)		-	12.51 ± 9.78
Total IgE (kU)		8.98 ± 2.36	223.68 ± 316.11*
Positive skin test		0 (0.0)	6 (31.6)
Blood WBCs (/µL)		6.31 ± 2.81	10.36 ± 3.23*	10.12 ± 3.86*
Blood eosinophils (%)		2.85 ± 2.42	1.83 ± 1.59	3.43 ± 4.48
Blood eosinophils (/µL)		0.16 ± 0.12	0.15 ± 0.11	0.28 ± 0.36
Blood neutrophils (%)		59.55 ± 9.74	66.6 ± 13.89	67.9 ± 13.23
Blood neutrophils (/µL)		3.81 ± 2.08	6.81 ± 3.59	7.1 ± 3.46*

Data are expressed as number (%), means ± standard error of the mean, or median (interquartile range).

FEV1, forced expiratory volume in 1 second; FVC, forced vital capacity; BMI, body mass index; PC20, the provocative concentration of methacholine required to decrease the FEV1 by 20%; IgE, immunoglobin E; WBC, white blood cell.

^*P < 0.05 compared to control subjects; ^†P < 0.05 compared to patients with stable asthma.

Enzyme-linked immunosorbent assay (ELISA)

The plasma CSN5 concentration was measured using ELISA (MyBioSource, Inc., San Diego, CA, USA). To compare results from different plates, the optical densities (ODs) of test samples were adjusted relative to the positive and negative controls provided in each kit. The mean OD of duplicate wells was calculated. The index value of each tested plasma was defined by the following formula: Index = (OD of Tested Plasma − OD of Negative Control) / (OD of Positive Control − OD of Negative Control) × 100. The minimum detection limit for CSN5 was set at 1.0 pg/mL according to the manufacturer’s recommendations.

Cell culture

HMVEC-L cells were cultured as previously described.²⁰ Cell culture media (EGM-2 BulletKit) were purchased from Lonza (Basel, Switzerland). HMVEC-L were maintained in EGM-2 BulletKit (Lonza) and maintained at 37°C in a 5% CO₂ humidified incubator.

Small interfering RNA (siRNA) and Der p 1 treatments

HMVEC-L were transfected with 50 nM CSN5 siRNA (Bionics, Seoul, Korea) using Lipofectamine 2000 (Invitrogen, Waltham, MA, USA). After incubating 50 nM CSN5 siRNA in 250 μL OPTI-MEM (Thermo Fisher Scientific, Rockford, IL, USA) for 5 minutes, the cells were mixed with 250 μL OPTI-MEM and 10 μL Lipofectamine and incubated for 20 minutes at room temperature. The mixture was added to each well and the medium changed to EBM-2 medium after 12 hours. At 48 hours after transfection, the CSN5-silenced cells (Table 2) were treated with 10 μg/mL Der p 1 at 4, 8, or 24 hours.

Table 2. siRNAs used in this study.

Gene name	Sequence (5′→3′)
CSN5	GAGCUGUUGUGGAAUAAAUTT

siRNA, small interfering RNA; CSN5, constitutive photomorphogenesis 9 signalosome subunit 5.

Animal exposure protocols

The Soonchunhyang University Institutional Animal Care and Use Committee approved the animal study (SCHBC-animal-2020-06). Female, 6-week-old BALB/c mice were sensitized by intraperitoneal (IP) injection on days 0 and 14 with 50 µg of grade V chicken egg OVA (Sigma-Aldrich, St. Louis, MO, USA) emulsified in 10 mg of hydroxyl aluminum plus 100 µL of Dulbecco’s phosphate-buffered saline (D-PBS). On days 28 to 30 and 44 to 77, all mice received intranasal (IN) challenge with 150 µg of grade III OVA (Sigma-Aldrich) in 50 µL of D-PBS. Control mice were sensitized and challenged with saline. Dexamethasone (Sigma-Aldrich) and PD-L1 inhibitor, nivolumab (Opdivo inj, Ono Pharmaceutical, Osaka, Japan) IP was performed 1 hour after each challenge with OVA. Airway responsiveness was measured in conscious mice 1 day before sacrifice. Mice were placed in a barometric plethysmography chamber (All Medicus, Anyang, Korea), and baseline readings were conducted for 3 minutes and averaged.

Bronchoalveolar lavage fluid (BALF) morphology analysis

To collect cell-free bronchoalveolar lavage fluid (BALF), BALF was centrifuged at 1,000 rpm within 15 minutes at 4°C. The cell pellets were used to stain with H&E. These cells were counted and classified as macrophages, eosinophils, neutrophils, or lymphocytes.

Western blot analysis

Protein extracts of mouse lung tissue were collected as previously described.²⁰ The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Table 3). Detection was performed using EzWestLumi plus western blot detection reagent (ATTO Corporation, Tokyo, Japan). The relative protein abundance was determined quantitatively by densitometric analysis after normalization to β-actin (Sigma-Aldrich).

Table 3. Antibodies used in Western blot analysis and immunohistochemistry.

Antibody	Host	Company	City/State	Country	Catalog number
CSN5	Mouse	Santa Cruz Biotechnologies	Santa Cruz, CA	USA	sc-13157
PD-L1	Mouse	Genetex	Irvine, CA	USA	66248-lg
NF-κB	Rabbit	Cell Signaling	Danvers, MA	USA	3034
p-NF-κB	Rabbit	Cell Signaling	Danvers, MA	USA	3033
IKKβ	Rabbit	Cell Signaling	Danvers, MA	USA	8943
p-IKKβ	Rabbit	Cell Signaling	Danvers, MA	USA	2697
IκBα	Rabbit	Cell Signaling	Danvers, MA	USA	9242
p-IκBα	Rabbit	Cell Signaling	Danvers, MA	USA	2859
IL-13	Rabbit	Abcam	Cambridge	UK	106732
IFN-γ	Rabbit	Abcam	Cambridge	UK	2166429
β-actin	Mouse	Sigma	Burlington, MA	USA	A1978

CSN5, constitutive photomorphogenesis 9 signalosome subunit 5; PD-L1, programmed death-ligand 1; NF-κB, nuclear factor-κB; p-, phospho-; IKKβ, inhibitor of κB kinase β; IL, interleukin; IFN, interferon.

Immunohistochemistry

The slides of mouse lung tissue were immunohistochemistry stained as previously described.²⁰ Non-specific binding was blocked using 1.5% horse serum before incubating with antibody (Table 3). The following day, the sections were incubated with an ABC Kit (Vector Laboratories, Burlingame, CA, USA). The color reaction was developed using a liquid DAB + substrate kit (Golden Bridge International Inc., Mukilteo, WA, USA).

PAS staining

The slides of mouse lung tissue were PAS stained as previously described.²¹ The goblet cell was quantified using ImageJ software (Version 1.8.0; National Institutes of Health, Bethesda, MD, USA).

Masson’s trichrome staining

The slides of mouse lung tissue were Masson’s trichrome stained as previously described.²¹ The positive trichrome-stained area was quantified using ImageJ software (National Institutes of Health).

Collagen assay

Collagen assays were performed according to the user manual of the Sircol collagen assay kit (Biocolor, Northern Ireland, UK). Specifically, 100 µL of the protein extract sample in lung tissue was mixed with 1 mL of Sircol dye for 30 minutes and centrifuged at 10,000 rpm for 5 minutes to precipitate the formed collagen-dye complex. After decanting the suspension, droplets were dissolved in 1 mL of Sircol alkali reagent and vortexed. A total of 100 µL of the acquired solution was read at 540 nm.

Statistical analysis

The data were analyzed using SPSS statistical software (ver. 22.0; IBM Corp., Armonk, NY, USA). All data are expressed as means ± standard error of the mean (SEM) or as medians with ranges. Group differences were compared by two-sample t-tests, Mann-Whitney tests, or Pearson’s χ² tests for normally distributed, skewed, and categorical data, respectively. Correlations between outcome measures were evaluated by calculating Pearson or Spearman correlation coefcients analysis. A P value < 0.05 was considered to indicate statistical significance.

RESULTS

Increased plasma CSN5 levels in patients with asthma and their association with the clinical variables in these patients

This study included 19 asthmatic patients (mean age, 63.2 years) and 10 healthy controls (mean age, 65.2 years); Table 1 displays the clinical characteristics. Forced expiratory volume in 1 second (FEV1) % predicted, forced vital capacity (FVC) % predicted, and FEV1/FVC ratio at initial measurements were lower values in asthmatic patients compared to those in healthy controls. Patients with asthma have higher total immunoglobin E levels, eosinophil counts, and neutrophil counts. However, body mass index did not differ significantly across the 2 groups. The average duration of asthma was 6.05 ± 4.65 years. The plasma CSN5 level (P < 0.001) was notably elevated in exacerbated asthmatics (n = 19) compared to healthy controls (n =10) or those with stable asthma (Fig. 1A). The CSN5 level was correlated with FVC % predicted (r = −0.455, P = 0.001), FEV_1% predicted (r = −0.496, P < 0.001) and FEV₁_FVC % predicted (r = −0.296, P = 0.041) in patients with asthma (Fig. 1B).

Fig. 1. Expression levels of CSN5 from patients with asthma. (A) The CSN5 level in healthy controls and patients with stable or exacerbated asthma. (B) Relationships of CSN5 with FVC % predicted (r = −0.455, P = 0.001), FEV1% predicted (r = −0.496, P < 0.001) and FEV1_FVC % predicted (r = −0.296, P = 0.041).

CSN5, constitutive photomorphogenesis 9 signalosome subunit 5; FVC, forced vital capacity; FEV1, forced expiratory volume in 1 second.

^*P < 0.05 vs. healthy controls; ^† p <0.05 vs. stable asthma patients.

Change in PD-L1 and signaling pathways following CSN5 silencing and Der p 1 treatment in HMVEC-L

To explore the underlying mechanism of house dust mite treatment and CSN5 silencing for PD-L1, we assessed the expression changes of CSN5 a deubiquitinating enzyme. To investigate whether CSN5 silencing affect PD-L1 and NF-κB expression, we performed western blotting on HMVEC-L treated with Der p 1 treatment (Fig. 2). CSN5 silencing in HMVEC-L resulted in a reduction of NF-κB protein expression at 4 hours and PD-L1 expression at 4, 8, and 24 hours following Der p 1 treatment (Fig. 2). NF-κB plays an essential role in inflammation by regulating the expression of numerous cytokines, transcription factors, and regulatory proteins. And these cells reduced expression of NF-κB at 4 hours, suggesting that CSN5 silencing has a more pronounced effect at earlier time points in HMVEC-L.

Fig. 2. ilencing of CSN5 affect PDL-1 and NF-κB expression in HMVEC-L. (A) Western blot analysis of the levels of CSN5, PD-L1, and signaling pathway proteins following Der p 1 treatment and CSN5 silencing in HMVEC-L. Densitometric analysis was performed using 3 western blots, and values were normalized to β-actin levels. (B-D) Densitometric data from the Western blots. Data are expressed as means ± standard deviation.

CSN5, constitutive photomorphogenesis 9 signalosome subunit 5; PD-L1, programmed death-ligand 1; Der p 1, Dermatophagoides pteronyssinus; HMVEC-L, human lung microvascular endothelial cell; siRNA, small interfering RNA; NF, nuclear factor.

^*P < 0.05 vs. controls; ^†P < 0.05 vs. Der p 1 treatment; ^‡P < 0.05 vs. CSN5 silencing.

AHR and cell differentials after PD-L1 inhibitor injection in asthmatic mice

The OVA-induced mouse model of asthma was established as shown in Fig. 3A. AHR is characterized by the narrowing of the airways in response to methacholine measurements, resulting in increased airway resistance. To delve into the fundamental mechanisms of PD-L1 inhibitors, we conducted AHR measurements to assess the extent of respiratory deterioration in each group.

Fig. 3. Effect of PDL-1 inhibitor on airway eosinophilic inflammation infiltration in OVA-induced asthma models. (A) PD-L1 inhibitor and dexamethasone treatment protocol in BALB/c mice. Eight BALB/c mice were exposed to saline (control) or OVA and treated with dexamethasone (5 mg/kg) or a PD-L1 inhibitor (3 mg/kg). (B) Increased AHR in OVA-sensitized/challenged mice but decreased AHR in PD-L1/dexamethasone-treated mice compared with control mice. Data are expressed as means ± standard error of the mean. (C) Total and differential cell counts in BALF. BALF was collected at 33 and 80 days. Total and differential cell counts were increased in OVA-sensitized/challenged mice but decreased after PD-L1 inhibitor and dexamethasone treatment compared with control mice. Data are expressed as means ± standard deviation.

PD-L1, programmed death-ligand 1; OVA, ovalbumin; AHR, airway hyperresponsiveness; BALF, bronchoalveolar lavage fluid; NC, normal control; IP, intraperitoneal; IN, intranasal.

^*P < 0.05 vs. day 33 NC; ^†P < 0.05 vs. day 80 NC; ^‡P < 0.05 vs. day 80 OVA-sensitized/challenged mice.

To determine whether PD-L1 inhibitor can reduce airway responsiveness and inflammation in an asthmatic mice model, we compared its effect with those of dexamethasone treatment. We assessed changes of airway responsiveness and inflammation in the asthmatic mice.

Airway obstruction, based on the enhanced pause (Penh), was greater in the OVA-sensitized/challenged groups and was reduced in mice treated with PD-L1 inhibitor or dexamethasone compared with the control group (Fig. 3B). Cell differentials, such as total cell, neutrophil, and eosinophil counts, were increased in the OVA-sensitized/challenged groups and decreased in mice treated with either PD-L1 inhibitor or dexamethasone compared with the control group (Fig. 3C).

Changes in airway inflammation, goblet cell hyperplasia, lung fibrosis, and CSN5 and cytokine expression after PD-L1 inhibitor injection in asthmatic mice

We investigated whether PD-L1 inhibitor and dexamethasone treatment could change inflammatory cells (Fig. 4A and B), Goblet cell hyperplasia (Fig. 4A and C), lung fibrosis (Fig. 4A and D), and CSN5 (Fig. 4A and E) in a murine asthma model. Lung fibrosis quantification was done using lung homogenate (Fig. 4F). Th2 and Th1 cytokines associated with airway inflammation were measured using lung homogenate in western blotting (Fig. 5).

Fig. 4. Effect of PDL-1 inhibitor on histological analysis in OVA-induced asthma models. (A) H&E, PAS, Masson’s trichrome, and CSN5 immunohistochemical staining of mouse lung tissue sections. (B-E) Inflammatory index and quantification of goblet cell hyperplasia and lung fibrosis based on Masson’s trichrome staining and CSN5 expression. (F) Quantification of collagen content. Data are expressed as means ± standard deviation.

H&E, hematoxylin and eosin; PAS, periodic acid–Schiff; CSN5, constitutive photomorphogenesis 9 signalosome subunit 5; NC, normal control; OVA, ovalbumin; PD-L1, programmed death-ligand 1.

^*P < 0.05 vs. day 33 NC; ^†P < 0.05 vs. day 80 NC; ^‡P < 0.05 vs. day 80 OVA-sensitized/challenged mice.

Fig. 5. Effect of PDL-1 inhibitor on cytokine in OVA-induced asthma models. (A) Western blot analysis of cytokine expression in mouse lungs after PD-L1 inhibitor or dexamethasone treatment. Western blot analysis of lung cytokine protein levels. Densitometric analysis was performed using three western blots, and values were normalized to β-actin levles. (B, C) Densitometric data from the Western blots. Data are expressed as means ± standard deviation.

PD-L1, programmed death-ligand 1; IL, interleukin; IFN, interferon; NC, normal control; OVA, ovalbumin.

^*P < 0.05 vs. day 33 NC; ^†P < 0.05 vs. day 80 NC; ^‡P < 0.05 vs. day 80 OVA-sensitized/challenged mice.

Airway inflammation, goblet cell hyperplasia, lung fibrosis, and the levels of CSN5 (Fig. 4A-E), collagen (Fig. 4F), IL-13 and interferon-γ (Fig. 5) were increased in the OVA-sensitized/challenged mice and decreased in mice treated with either PD-L1 inhibitor or dexamethasone compared with the control group.

Changes in signaling pathways after PD-L1 inhibitor injection in asthmatic mice

The NF-κB coordinates the activation of genes associated with inflammation and airway remodeling in asthma. The function of NF-κB is precisely controlled by IκB-α, a process that can be influenced by genetic variations in the IκB-α gene. NF-kB family of transcription factors has been extensively described as a central regulator of both inflammation and tumor-proliferative and cell-survival pathways.

In addition to other NF-κB proteins, inhibitor of κB kinase β (IKKβ) was also present.²²,²³,²⁴ The tumor-suppressing properties of death associated protein kinase 1 through downregulation of IKKβ/CSN5/PD-L1 axis in gastric cancer were further confirmed in vivo.²⁵

To delineate the role of signaling way such as IKKβ, phospho- (p-)IKKβ, IκBα, p-IκBα, and p-NF-κB in asthmatic mice, we determined the expression levels of proteins of signaling pathway using western blotting. Protein levels of lung PD-L1, p-NF-κB, p-IκBα, and p-IKKβ increased at both 33 and 80 days in OVA-sensitized/challenged mice relative to the control group. Treatment with a PD-L1 inhibitor and dexamethasone resulted in a reduction of PD-L1, CSN5, p-NF-κB, p-IκBα, and p-IKKβ protein levels compared with levels in OVA-sensitized/challenged mice (Fig. 6). Lung IκBα and IKKβ protein levels decreased in OVA-sensitized/challenged mice at 33 and 80 days in comparison to control mice; however, treatment with a PD-L1 inhibitor reversed these changes (Fig. 6). In OVA-sensitized/challenged mice, the protein levels of lung PD-L1, p-NF-κB, p-IκBα, and p-IKKβ were elevated at both 33 and 80 days compared to control mice. Treatment with a PD-L1 inhibitor and dexamethasone resulted in a reduction of PD-L1, CSN5, p-NF-κB, p-IκBα, and p-IKKβ protein levels when compared to levels observed in OVA-sensitized/challenged mice (Fig. 6).

Fig. 6. -Effect of PDL-1 inhibitor on CSN5 and signaling pathway in OVA-induced asthma models. (A) Western blot analysis showing the changes in signaling pathway protein and CSN5 levels in mouse lungs after PD-L1 inhibitor or dexamethasone treatment. (B-J) Quantification of protein expression. Densitometric analysis was performed using three western blots, and values were normalized to β-actin levels. Data are expressed as means ± standard deviation.

CSN5, constitutive photomorphogenesis 9 signalosome subunit 5; PD-L1, programmed death-ligand 1; NC, normal control; OVA, ovalbumin; IKKβ, inhibitor of κB kinase β; p-, phospho-; NF-κB, nuclear factor-κB.

^*P < 0.05 vs. day 33 NC; ^†P < 0.05 vs. day 80 NC; ^‡P < 0.05 vs. day 80 OVA.

DISCUSSION

The plasma CSN5 level was higher in patients with exacerbated asthma and was correlated with pulmonary function in asthma patients. The levels of CSN5 and signaling pathway proteins were altered by PD-L1 inhibitor treatment in cells and mouse lungs, indicating that CSN5 coordinates with PD-L1 in asthma and may be a possible target for asthma therapy.

The CSN5 functions in ubiquitin-mediated protein degradation. Dysregulation of CSN5 may dramatically affect various cellular functions, such as programmed cell death, angiogenesis, and signal transduction.²⁶ Although increasing evidence has demonstrated that CSN5 is highly expressed in various human malignancies and is commonly associated with a dismal prognosis, the mechanisms underlying its function are poorly understood.²⁶ The CSN is an evolutionarily conserved complex consisting of eight subunits (CSN1–CSN8). CSN5 is an extremely important subunit of the CSN that functions as an oncogene in NSCLC and holds promise as a potential diagnostic and therapeutic target for the disease.²⁷,²⁸ Among the CSN subunits, CSN5 and its dimerization partner, CSN6, are the only 2 Mpr1/Pad1/N-terminal domain-containing subunits. They play crucial roles in various biological functions, such as cell cycle progression, protein stability, and signal transduction.²⁷,²⁸ In the present study, CSN6 was not investigated.^-Further studies are needed to clarify the role of CSN6 in asthma.

Currently there are no data on the role of CSN5 in asthma pathogenesis. In this study, CSN5 expression was increased in the lungs of asthmatic mice and in HMVEC-L treated with house dust mites. Additionally, the plasma CSN5 level was higher in patients experiencing exacerbated asthma compared to those with stable asthma. This implies that CSN5 is implicated in asthma and serves as a marker of asthma exacerbation.

The CSN regulates cellular proliferation and apoptosis, affecting a series of pathways as well as regulating genomic instability and DNA damage/repair.⁹,²⁹ CSN subunits have been widely investigated in various experimental systems and shown to be involved in cell cycle regulation, immune cell homeostasis, and the immune response.³⁰ CSN5, a key component of the signalosome complex, has been implicated in numerous signaling pathways; however, its mechanism of action is poorly understood in asthma. In this study, p-IκBα, p-IKKβ, and p-NF-κB protein levels were altered in mouse lungs following PD-L1 inhibitor treatment, indicating that CSN5 interacts with various signaling pathways to play an important role in airway inflammation.

Asthma is a significant respiratory disease characterized by chronic airway inflammation and remodeling.³¹ Previously, the treatment of severe asthma included administering elevated doses of inhaled corticosteroids combined with prolonged treatment with β-agonists, theophylline, leukotriene antagonists, or anticholinergic agents. However, the benefits of these medications were often limited.³² While recent agents targeting Th2 asthma endotypes have led to improved therapeutic outcomes in asthma, the diverse mechanisms of these drugs are still poorly understood.³²,³³ Biologics and most small molecules have been shown to alter the strength, quality, or amount of particular asthma endotypes, targeting PD-L1/PD-L2, GATA-3, and CD38, thymic stromal lymphopoietin, tryptase, Janus kinase, for the therapy and control of Th2 type asthma.³³

PD-1 is an inhibitory molecule expressed on the surface of activated T-cells, and it binds to PD-L1 and PD-L2 ligands.³⁴ Metabolic reprogramming occurs in activated T cells affected by PD-1 signaling.³⁵ The identification of immune checkpoint proteins, such as PD-1/PD-L1 and cytotoxic T-lymphocyte associated protein-4, represents a significant discovery in the field of cancer immunotherapy.³⁵ PD-L1 can be targeted by both PD-1-specific antibodies and monoclonal antibodies. Therefore, humanized monoclonal antibodies targeting these immune checkpoint proteins have been successfully used in patients with various carcinoma, head and neck cancers, and NSCLC.³⁶,³⁷,³⁸

PD-L1 and PD-L2 play important but opposing roles in modulating and polarizing T-cell functions in airway hyperreactivity. Whereas the severity of asthma is significantly enhanced in absence of PD-L2, PD-L1 deficiency resulted in reduced AHR and only minimal inflammation.¹⁸

Currently, there are no data regarding the effects of PD-L1 inhibitors in asthma. However, it has been shown that various signaling pathways are involved in airway diseases.³⁹,⁴⁰,⁴¹,⁴²,⁴³ In this study, the potential mechanisms associated with the effects of CSN5 on PD-1 blockade therapy in asthma were evaluated. Treatment with a PD-L1 inhibitor decreased airway obstruction and inflammation, goblet cell hyperplasia, lung fibrosis, and levels of CSN5 and IL-13 in asthmatic mice. In addition, PD-L1 inhibitor treatment reduced the lung PD-L1 level in signaling pathways such as p-NF-κB, p-IκBα, and p-IKKβ. These findings indicate that CSN5 and PD-L1 participate together in airway inflammation via signaling pathways in asthmatic mice (Fig. 7).

Fig. 7. Summary of study. CSN5 and PD-L1 participate in airway inflammation via signaling pathways in asthma.

CSN5, constitutive photomorphogenesis 9 signalosome subunit 5; PD-L1, programmed death-ligand 1; IKKβ, inhibitor of κB kinase β; NF, nuclear factor; PD-1, programmed cell death protein 1; IFN, interferon; IL, interleukin.

This study has several limitations. First, the small sample size of asthma patients included was a constraint. Second, while the study focused on the involvement of asthma through PD-L1 inhibitor and CSN gene inhibition, it did not establish a model for PD-1 inhibition. Thirdly, further studies need to be conducted to investigate the effects of PD-L1 and CSN5-related signaling on airway epithelial cells and to clarify their roles in the initiation of allergen activation responses after entry into the airway.

In conclusion, the plasma CSN5 level was higher in exacerbated asthmatics than in healthy controls or stable asthmatics. In addition, CSN5 levels associated with FEV1 in patients with asthma and with various cellular signaling pathways in mouse lungs. Furthermore, PD-L1 inhibitor decreased the CSN5 level and altered the associated signaling pathways, indicating that CSN5 cooperates with PD-L1 in asthma. This study suggests that CSN5 could be a new marker of asthma exacerbation as well as a possible therapeutic target.