The expression and function of programmed death-ligand 1 and related cytokines in neutrophilic asthma

Introduction

Bronchial asthma is a common chronic respiratory disease. It is defined by a history of respiratory symptoms that vary over time and in intensity, and a variable expiratory airflow limitation (1,2). Asthma effects approximately 300 million individuals worldwide, and its prevalence is increasing in many developing countries (3). There are approximately 30 million asthmatic patients in China, of whom 1/3 are children (4). The prevalence of asthma in children is increasing (5).

The pathogenesis of asthma is complex. It has been shown that skewed T helper cell type 2 (Th2) cell responses and decreased T helper cell type 1 (Th1) cell responses are important mechanisms mediating asthma. Th2 cells release immunoglobulin E (IgE) and interleukins (ILs), including IL-4, IL-5, and IL-13, to activate eosinophils, mast cells, innate lymphoid cells, and other inflammation cells. They also reduce Th1 cell immune responses and decrease interferon gamma (IFN-γ) secretion, which weaken the inhibition of the Th2 immune response and cause chronic airway inflammation (6,7). Notably, approximately 50% of asthma patients do not have skewed Th2 cell immune responses. These patients do not display significant eosinophilic airway inflammation (8). Recent studies have classified asthma into two broad types: (I) eosinophilic asthma (EA); and (II) non-EA (NEA). Neutrophilic asthma (NA) is an important inflammatory phenotype of NEA. NA is associated with disease severity, airflow limitation, and steroid resistance. Its underlying mechanism is still uncertain.

T helper cell type 17 (Th17) cells represent a distinct subset of inflammatory cells characterized by a high level of production of IL-17. Th17 cells have been implicated in the immunopathology of both asthma and chronic obstructive pulmonary disease. Th17 effector cells recruit neutrophils into the airway (9,10). Th17 cells and their-related cytokines (e.g., IL-17, IL-21, and IL-22) contribute to the development of severe asthma or refractory asthma. IL-17 family cytokines also target innate immune cells and epithelial cells to produce granulocyte colony-stimulating factor (G-CSF) and IL-8 (11,12). They can also recruit and activate neutrophilic granulocytes to the airway to mediate severe inflammation (13).

Lymphocyte activation and differentiation are modulated by co-stimulatory molecules provided by antigen presenting cells (APCs). Programmed death-ligand 1 (PD-L1) is expressed on resting B cells, T cells, macrophages, and dendritic cells (DCs) (14). PD-L1 regulates T cell-mediated immune responses by acting as both a T cell co-stimulator and co-inhibitor (15,16). Recent studies have shown that PD-L1 has an important function in allergies and asthma, but the data on the function of PD-L1 and related cytokines of Th cells in NA have been limited and the definitive mechanism underlying PD-L1 in NA is unclear. we found that PD-L1 and related cytokines were linked with NA in both murine models and clinical research. We sought to study the expression levels of soluble PD-L1 (sPD-L1) in the bronchoalveolar lavage fluid (BALF) of children with NA, and analyze its correlation with the expression of Th1, Th2, and Th17-type cytokines. Further, using neutralizing antibody (Ab) specific to PD-L1 and an experimental NA mouse model, we examined the immune mechanism of PD-L1 in NA, and its relationship to Th1, Th2, and Th17 cells. Some patients with NA respond poorly to inhaled high dose of corticosteroid treatment, PD-L1 Ab might be a useful immunotherapeutic treatment of NA. We present the following article in accordance with the ARRIVE reporting checklist (available at https://dx.doi.org/10.21037/atm-21-5648).

Methods

Human subjects

Samples from 26 asthma patients were collected from the Department of Respiratory Medicine of the Children’s Hospital of Soochow University from October 2015 to November 2016. The diagnosis and severity of an asthma attack were determined according to the Global Initiative for Asthma guidelines (17). The patients were allocated into a NA group or non-NA group based on the amounts of neutrophils in their BALF. Each group comprised 13 patients. Patients with ≥48% neutrophils and ≤1% eosinophils in their BALF were diagnosed with NA, while the others were diagnosed with non-NA. Fifteen children who had been hospitalized for bronchial foreign body treatment were enrolled in the control group. The inhalation time was <1 day. The patients had to have no personal or family allergic disease history or history of lung infection within the last 6 months. The study was approved by the Institutional Human Ethics Committee of the Children’s Hospital of Soochow University (No. 2014009), and written consent was obtained from the parent/guardian of each child participating in this study. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013).

Specimen collection

The BALF was collected from the asthmatic children by bronchoalveolar lavage within 24 to 48 hours of hospitalization. To qualify, each specimen had to meet the following criteria: the percentage of recovery of the BALF had to be >40%, and red blood cells had to be <20%. The BALF of the control group was collected in a similar manner.

Mice

A total of 22 specific-pathogen-free BALB/c mice (females, aged 6–8 weeks old, weighing 18–20 g) were purchased from the JOINN Laboratories, Suzhou, China. The mice were housed in barrier cages under controlled environmental conditions (12/12 h light/dark cycle, 55%±5% humidity, 23±1 °C). They had free access to pelleted food and pure water. Animal experiments were performed under a project license (No. SUDA20200510A02) granted by the Ethics Committee for Animal Use of Soochow University, in compliance with European Community Guidelines for the care and use of animals (18).

Experimental groups

The mice were randomly divided into the following four groups using a random number table: the NA group (n=6), the phosphate-buffered saline (PBS) control group (n=4), the immunoglobulin G (IgG)-treatment NA group (n=6), and the PD-L1 Ab-treated NA group (n=6). The NA group, the IgG-treatment NA group, and the PD-L1 Ab-treated NA group were sensitized with 0.2 mL ovalbumin (OVA) (Sigma-Aldrich, Los Angeles, California), which contained 20 µg of OVA and 10 µg of lipopolysaccharides (LPS) in PBS, and was intraperitoneally injected on days 0, 7, and 14. The IgG-treated NA mice and the PD-L1 Ab-treated NA mice were intraperitoneally injected with 20 µg of IgG and B7-H1 Ab on days 0, 3, 7, 10, and 14 after the OVA injection. The PBS control mice were intraperitoneally injected with PBS on the same days and inhaled PBS in a similar manner. From day 21, these mice were challenged with aerosolized 1% OVA daily for 7 consecutive days (see Figure 1).

Figure 1 Experimental timeline. NA group, PBS control group, IgG-treated NA group and PD-L1 Ab-treated NA group. NA, neutrophilic asthma; PBS, phosphate-buffered saline; IgG, immunoglobulin G; PD-L1, programmed death-ligand 1; Ab, antibody; OVA, ovalbumin; LPS, lipopolysaccharides.

Specimen collection

Twenty-four hours after the last inhalation of OVA, the mice were anesthetized and then killed. Blood was retro-orbitally collected from the mice, and anticoagulated with ethylenediaminetetraacetic acid. The right lung of each mouse was ligated and removed for histological and flow cytometric analyses. The left principal bronchus was cannulated with a polyethylene tube. A total of 1.5 mL of BALF was collected.

Lung histology of mice model

The right lungs were first perfused with 4% paraformaldehyde in 0.1 M PBS, fixed with formalin, and embedded with paraffin after dehydration. The 3-µm paraffin sections were stained with hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) to measure neutrophil infiltration and mucus production, respectively. Other paraffin sections were incubated with the rat anti-PD-L1 monoclonal Ab (mAb) at 4 °C overnight after antigen repair. After incubation with an anti-rat IgG Ab conjugated with horseradish peroxidase, the specimens were developed using the diaminobenzidine coloration method.

Enzyme-linked immunoassay (ELISA) for cytokines

The concentrations of sPD-L1, IL-4, IL-6, IFN-γ, IL-17, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the BALF supernatants collected from patients were measured using ELISA kits (R&D, USA) in accordance with the manufacturer’s instructions. The concentrations of the proinflammatory cytokines of IL-4, IL-6, IL-17, IFN-γ, tumor necrosis factor alpha (TNF-α), and G-CSF in the plasma and the BALF of the mice were assessed using ELISA kits (R&D, USA) in accordance with the manufacturer’s instructions.

BALF cytology

The pellets of BALF were resuspended in 100 µL PBS and pipetted onto glass slides to count the total cell numbers. The cell differential count was performed using Wright-Giemsa staining. Four hundred cells were counted under a light microscope. The results are expressed as the total cell number ×10⁴/mL.

Flow cytometric analysis

The cells were freshly isolated from the mice lungs following digestion with Collagenase type I (Worthington Biochemical, USA) and DNase I (Worthington Biochemical, USA) for 90 min at 37 °C. Single-cell suspension was collected and incubated with Erythrocyte lysate (Biolegend, USA) for 5 min at 4 °C. After incubation, the cells were washed twice with PBS, and incubated with the anti-mouse CD16/32 Fc block (Biolegend, USA) at 25 °C for 15 min. The following fluorochrome-labeled Abs were obtained from Biolegend, USA: FITC-CD45, PE/Cy7-CD11C, APC/Cy7-CD11b, APC-F4/80, and PB-MHC-II. The cells were fixed with antibodies, and each sample was allocated to the experimental group or the control group. The experimental group cells and control group cells were incubated with PE-PD-L1 and PE-Rat IgG2a, respectively at 4 °C for 20–30 min. The cells were washed twice with PBS, resuspended, and analyzed by flow cytometry (Beckman Coulter Gallios, USA).

Statistical analysis

Measurement data with a normal distribution are expressed as mean ± standard deviation (SD). The chi-square test was applied to the numerical data. A one-way or multiple analysis of variance (ANOVA) was used to compare the cell classifications and cytokines among different groups. Spearman’s correlation analysis was used to examine the correlation of cytokines in the BALF of NA patients. The statistical analysis was performed by SPSS 18.0 software (SPSS Inc., Chicago, IL, USA) using the rank-sum test and a one-way ANOVA. Values of P<0.05 were considered statistically significant.

Results

Medical history of cases

All 26 asthma patients had symptoms of coughing and wheezing, and 10 of the patients had fever (38.5%), 6 had shortness of breath (23.1%), and 3 had dyspnea (11.5%). There was no significant difference in the age and sex ratio among the three groups (P>0.05; see Table 1).

BALF cell classification

The percentages of lymphocytes and neutrophils in the BALF of NA patients were significantly higher than those of non-NA and control patients (P>0.05). The percentage of eosinophils in the BALF of non-NA patients was significantly higher than that of NA and control patients (P<0.05; see Table 2).

Comparison of cytokines in the BALF of the three groups

NA patients had significantly higher levels of sPD-L1 (27.61±33.35 ng/L), IL-6 (24.19±20.43 ng/L), and IL-17 (17.12±11.46 ng/L) in their BALF than non-NA and control patients (P<0.05). Additionally, the NA patients had significantly elevated BLAF GM-CSF compared to the non-NA and control patients (P<0.05). Conversely, non-NA patients had higher IL-4 concentrations (24.42±36.96 ng/L) but lower IFN-γ (34.42±27.37 ng/L) than NA and control patients (P<0.05). There was no significant difference in sPD-L1, IL-6, and IL-17 in the BALF between non-NA and control patients, and there was no significant difference in IL-4 and IFN-γ between NA and control patients (see Figure 2). These data suggest that sPD-L1 is associated with increases of IL-17 and IL-6 in the BLAF of NA patients.

Figure 2 Concentrations of cytokines (A) sPD-L1, (B) IL-4, (C) IL-6, (D) IFN-γ, (E) IL-17, and (F) GM-CSF in the BALF of the three groups. sPD-L1, soluble programmed death-ligand 1; IL, interleukin; IFN-γ, interferon gamma; GM-CSF, granulocyte-macrophage colony-stimulating factor; BALF, bronchoalveolar lavage fluid; NA, neutrophilic asthma.

The linear correlation analysis of cytokines in the BALF of NA patients

To further confirm these results, we performed a linear regression analysis to examine linear correlations between the concentration of sPD-L1 and GM-CSF, IL-6, IFN-γ, and IL-17. In the NA patients, the concentration of sPD-L1 was significantly correlated with the level of GM-CSF, IL-6, IFN-γ, and IL-17 in the BALF (r=0.879, P<0.001; r=0.718, P=0.006; r=0.791, P=0.001; r=0.726, P=0.005), but was not correlated with IL-4 (r=−0.349, P=0.242). IL-17 level was positively correlated with IL-6 and GM-CSF in the BALF (r=0.890, P<0.001; r=0.817, P=0.001), but was not correlated with IL-4 (r=−0.029, P=0.924; see Figure 3).

Figure 3 Linear correlation analysis of the NA groups. (A) Linear correlation between the sPD-L1 and GM-CSF; (B) linear correlation between the sPD-L1 and IL-6; (C) linear correlation between the sPD-L1 and IFN-γ; (D) linear correlation between the sPD-L1 and IL-17; (E) linear correlation between the IL-17 and IL-6; (F) linear correlation between the IL-17 and GM-CSF. NA, neutrophilic asthma; GM-CSF, granulocyte-macrophage colony-stimulating factor; sPD-L1, soluble programmed death-ligand 1; IL, interleukin; IFN-γ, interferon gamma.

The syndromes in the mouse model

To examine the role of PD-L1 in mediating NA, we used an asthmatic mouse model. Asthmatic syndromes were observed in the mice induced by OVA after a 10–15 min aerosol challenge on days 22 to 28. The asthmatic syndromes of the NA mice and the IgG-treated NA mice, such as cough, tachypnea, nodding breathing, and urine incontinence, were significantly more severe than those of the PBS control mice. The clinical symptoms of the NA treated mice with PD-L1 Ab were markedly alleviated compared to those of the IgG-treated mice. The semi-quantitative score analysis showed that the mean [interquartile range] of the NA mice of 2 [2–2] was significantly higher than that of the PBS control mice of 0 [0–1] (P<0.001). The semi-quantitative score of the PD-L1 Ab-treated NA group of 1 [1–2] was significantly lower than that of the NA asthma group (P=0.003).

Cytology in the BALF in the mouse model

The recovery ranged from 81% to 98% of the BALF introduced. As Table 3 shows, the NA mice and the IgG-treated NA mice showed hypercellularity compared to the PBS control mice and the PD-L1 Ab-treated NA mice. The total numbers of cells and the percentages of neutrophils were significantly higher in the NA mice and the IgG-treated NA mice than the PBS control mice and the PD-L1 Ab-treated NA mice. Additionally, the total number of cells and the percentage of neutrophils in the PD-L1 Ab-treated NA mice were significantly higher than those in the PBS control mice. There was no significant difference in eosinophils among the four groups of mice.

Flow cytometric analysis of PD-L1 in lungs

The percentages of PD-L1-positive cells in the lungs were significantly higher in the NA mice and the IgG-treated NA mice than the PD-L1 Ab-treated NA mice (P<0.05). No significant difference was found between the PBS control mice and the PD-L1 Ab-treated NA mice (see Figure 4). These data suggest that PD-L1 plays an important role in mediating the development of NA.

Figure 4 Percentage of PD-L1 in the lungs of different groups. PD-L1, programmed death-ligand 1; NA, neutrophilic asthma; PBS, phosphate-buffered saline; IgG, immunoglobulin G; Ab, antibody.

Comparison of cytokines in the plasma and BALF of the experimental mice

As Table 4 shows, the production of IL-17, IL-4, TNF-α, and G-CSF was significantly more downregulated in the blood plasma of the PD-L1 Ab-treated NA mice than the NA mice and the IgG-treated NA mice (P<0.05). The PD-L1 Ab-treated NA mice had significantly higher concentrations of IL-17, IL-4, TNF-α, and G-CSF in their plasma than the PBS control mice (P<0.05). Notably, the PBS control mice had higher concentrations of IFN-γ in their plasma than the other three groups (P<0.05). There was no significant difference in the concentrations of IL-6 in the plasma among the four groups (see Table 3). Thus, the results suggest that PD-L1 Ab treatment decreases airway inflammation by restricting Th2 and Th17-cell responses.

We also examined the cytokines in the BALF of the experimental mice. The NA mice had higher levels of IL-17 (72.59±9.50 pg/mL), IL-4 (32.26±4.73 pg/mL), G-CSF (240.97±184.04 pg/mL), IL-6 (13.55±0.58 pg/mL), and TNF-α (309.43±53.15 pg/mL) (P<0.05; see Figure 5) than the PBS control mice. The levels of IL-17, IL-6, and G-CSF were significantly lower in the PD-L1 Ab-treated mice than the NA mice (P<0.05, Figure 5). Further, the production of IL-4 (28.81±1.99 pg/mL), G-CSF (205.47±13.51 pg/mL), and TNF-α (286.43±34.65 pg/mL) was significantly more decreased in the BALF of the PD-L1 Ab-treated mice than the BALF of the untreated NA mice (see Figure 5). However, PD-L1 Ab treatment did not affect the level of IFN-γ production in the BLAF (see Figure 5). There was no significant difference in the production of IL-6 and IL-17 between the PBS control mice and the PD-L1 Ab-treated NA mice (see Figure 5). These findings suggest that PD-L1 increases IL-17, IL-4, and G-CSF in the BLAF of the NA mice, thereby contributing to airway inflammation.

Figure 5 Concentrations of cytokines (A) IL-17, (B) IFN-γ, (C) IL-4, (D) IL-6, (E) TNF-α, and (F) G-CSF in the BALF of the different mice groups. IL, interleukin; IFN-γ, interferon gamma; TNF-α, tumor necrosis factor alpha; G-CSF, granulocyte colony-stimulating factor; BALF, bronchoalveolar lavage fluid; NA, neutrophilic asthma; PBS, phosphate-buffered saline; IgG, immunoglobulin G; Ab, antibody.

PD-L1 Ab treatment reduced pulmonary inflammation and suppressed mucus production

Finally, to investigate the effects of PD-L1 Ab treatment on airway inflammation and mucus production in the lung tissue, we examined pulmonary pathology using H&E and PAS staining. The mice treated with OVA + LPS or with OVA + LPS + IgG exhibited more severe airway inflammation, mucus cell metaplasia, and had higher percentages of neutrophils and lymphocytes than those treated with PD-L1 Ab (see Figure 6A,6B). As Figure 6A shows, the neutrophil infiltration in the lung tissues of mice treated with PD-L1 Ab was significantly reduced. Notably, PD-L1 Ab treatment also significantly decreased mucus production in the lung tissue of the mice (see Figure 6B). The immunohistochemical studies showed that PD-L1-positive cells were increased in the NA mice and the IgG-treated NA mice (see Figure 6C). As Figure 6C shows, PD-L1 was decreased in the lung tissue of the PD-L1 Ab-treated mice.

Figure 6 Histological Analysis of Lung Sections of different groups. (A) The lung tissues of different groups were stained with H&E (original magnification ×400, scale bars =50 µm); (B) the lung tissues of different groups were stained with PAS to examine mucous secretion (original magnification ×400, scale bars =50 µm); (C) PD-L1-positive cells in various groups by immunohistochemistry detection (original magnification ×200, scale bars =100 µm). a: NA mice; b: PBS control mice; c: IgG-treated NA mice; d: PD-L1 Ab-treated NA mice. H&E, hematoxylin and eosin; PAS, periodic acid-Schiff; PD-L1, programmed death-ligand 1; NA, neutrophilic asthma; PBS, phosphate-buffered saline; IgG, immunoglobulin G; Ab, antibody.

Discussion

Immunological factors are thought to play important roles in mediating asthma. Research has shown that an imbalance of Th1/Th2 cells may be a major contributor to the development of asthma (19,20). Th2 cells release IL-4 to further enhance the differentiation of CD4⁺ Th precursors into Th2-like cells. Th2 cells also promote B cell responses to produce IgE. Together, the Th2 cell responses and B cell-produced IgE combine to recruit eosinophils and other inflammatory cells to the airway. This process leads to immediate hypersensitivity and airway inflammation, which causes bronchial hyperresponsiveness and airway structural remodeling (21-23). Th1-derived cytokine IFN-γ, which can induce the activation of macrophages and generate immunoglobulin, is mainly characterized by its cytotoxic effects (24). IFN-γ can antagonize IL-4 by repressing Th2 cell responses and IgE production. Augmenting Th1 immune responses may effectively reduce chronic inflammatory reactions mediated by Th2 cell responses (25). In the present study, we found that IL-4 in the BALF of non-NA patients was significantly higher than that of NA and control patients. Notably, IFN-γ in the BALF of non-NA patients was significantly lower than that of NA and control patients. These findings support previous results that non-NA patients, who primarily suffered from EA, had imbalanced Th1/Th2 responses, but highly activated Th2 cell responses. In our clinical study, we also found that there was no significant difference in the IL-4 and IFN-γ in the BALF between NA patients and control patients. This implies that the imbalance of Th1/Th2 cells may not be a major contributor to the development of NA.

Previous studies have shown that an imbalance between Th17/Treg and/or Th17/Th2 cells is related to NA, severe asthma, and refractory asthma (2,26). In severe asthmatic patients, imbalanced Th17/Treg or Th1/Th17 responses recruit and activate neutrophils into the airway (26-28). Th1 inflammatory cytokines are also effect in NA. IFN-γ, a signature Th1 cytokine, and its level was reported to be increased in BALF from severe asthmatic patients (20). Liu et al. (29) uncovered that Th2/Th17-low asthma that was associated with NA. And they also found that the levels of IL-1 α, IL-6, IL-8, G-CSF, and GM-CSF increased in the group of Th2/Th17-low patients. Type 2 cytokine suppression improved TH17 responses in a preclinical model of allergen-induced asthma. Th17 cells and neutrophilic inflammation increased in the lung by neutralization of IL-4 and/or IL-13 (11). Th17 cells mainly produce IL-17A, IL-17F, IL-6, IL-22, GM-CSF, and other cytokines. They cause potent proinflammation responses by recruiting and activating neutrophils (13). High level of IL-17A in serum is an independent risk factor for severe asthma that impacts neutrophil- airway smooth muscle remodeling and hypercontractility (30). IL-17 is a signature cytokine of Th17 cells that promotes IL-8 production by airway epithelial cells and smooth muscle cells. IL-8 is a chemokine which recruit neutrophils and granulocytes. As a neutrophil-specific chemotactic factor, IL-8 can recruit neutrophils into the airway. Additionally, IL-17 can stimulate airway cells to produce high levels of IL-6 and G-CSF, which stimulate neutrophil development and granulopoiesis to induce neutrophilic responses (31-33). Th1, Th2 and Th17 immunity may cooperate to enhance asthma severity or interact to suppress the activity of on another (30).

Clinical studies have shown that the levels of IL-17A and Thl7 cells in the sputum of NA patients are significantly higher than those in the sputum of EA patients, and are correlated with the percentage of neutrophils in sputum, which suggests that Thl7 cells mediate airway inflammation by IL-17A (34). Zhao et al. developed a mouse model by exposing OVA-induced asthmatic mice to LPS (35). Neutrophil-predominant airway inflammation was observed in vivo in these mice with increases of airway hyperresponsiveness (AHR) and increases of Th17 cells and IL-17A levels in the BALF. In our clinical study, we found that NA patients had significantly higher levels of sPD-L1, IL-6, IL-17, and G-CSF than non-NA and control patients, and lower IL-4 levels than non-NA patients. In NA patients, the level of IL-17 was positively correlated with G-CSF and IL-6. Thus, IL-17 may induce the secretion of G-CSF and IL-6 and serve as a mediator that induces NA. These data suggest that augmented Th17 cell responses are associated with neutrophilic inflammation and asthma. These responses may be a major mechanism in the development of NA in patients.

Eisenbarth et al. (36) found that in the mouse model, high dose of LPS during intranasal OVA priming resulted in Th1 cells inflammatory response dominated by neutrophils. And the level of IFN-γ also increased in high dose LPS-exposed mice. Lajoie et al. (37) found that IL-17A and Th2 cytokine levels were elevated in a severe asthmatic murine model. This suggests that Th17 and Th2 cells are related to severe asthmatic inflammation. IL-17A may increase neutrophilic inflammation. Cosmi et al. (31) described a new type of memory Th17/Th2 cells in the circulating blood of humans that appear to be significantly increased in patients with chronic severe asthma. These Th17/Th2 lymphocytes may originate from allergen-specific T lymphocytes on IL-4 stimulation. Cosmi et al. hypothesized that an IL-4-rich microenvironment may encourage allergen-specific Th17 lymphocytes to switch to Th17/Th2 phenotypes. Preclinical studies have shown that exposing young and adult mice to a combination of diesel exhaust particle (DEP) and house dust mite (HDM) provokes an exaggerated Th2 and mixed Th2/Th17 cell-mediated inflammatory response in the airways (38). In our study, we found that after treatment with OVA + LPS or with OVA + LPS + IgG, the lungs of the mice displayed more severe airway inflammation. The total number of cells and the percentage of neutrophils were significantly. We also found that the levels of IL-4, IL-17, G-CSF, and TNF-α in the plasma and the BALF of the NA mice were significantly higher than those in the PBS control mice, but the level of IFN-γ in the plasma and the BALF was lower than that in the control group. Additionally, despite blocking the PD-L1 signaling pathway, there was no significant difference in the eosinophil levels in the BALF between the groups. Combined with others’ observations, our findings indicate that dysregulated T cell responses, including Th1, Th2, and Th17 cell responses, are associated with the development of NA. Further, a decrease in Th1 cells and the hyper-activation of Th2/Th17 responses may lead to neutrophil aggregation.

It is well known that the B7 family plays a major role in providing co-stimulatory or co-inhibitory signals for T cells (39). PD-L1 has important regulatory functions in T cell-mediated immune responses and innate immune inflammatory responses. The interaction between PD-1 and PD-L1 leads to protection against self-reactivity. PD-L1 plays a critical immunoregulatory role in the chronicity of autoimmune diseases, tumors, graft rejection, and infectious diseases (40,41). The mechanism of PD-1 and PD-L1 inhibitory signaling also plays a crucial role in neurodegenerative diseases. PD-1/PD-L1 interaction plays protective role in autoimmunity diseases such as multiple sclerosis and rheumatoid arthritis (42). However, its express high levels in tumor cells and result in T cell suppression by interfering with T cell receptor signal transduction, which protect cancer cells from attack of immune response (43). Studies have also shown that PD-L1/programmed death-1 (PD-1) inhibits the proliferation and activation of CD4 T and CD8 T cells and decreases the levels of IL-2 and IFN-γ (44,45). Conversely, some studies have suggested that naïve T cells are primed or trapped by co-stimulatory PD-L1 signals. Singh et al. (14) found that combining PD-1 from invariant natural killer T (iNKT) cells with PD-L1 from DC greatly enhanced levels of IL-4 production and decreased levels of IFN-γ. Akbari et al. (46) found that a significantly greater reduction in eosinophils and macrophages in the BALF led to a reduction in AHR of the PD-L1^−/− mice compared to the WT BALB/c mice after OVA sensitization and challenge. Further, PAS staining showed that the PD-L1^−/− mice had less cell infiltration and did not produce mucus. Mcalees et al. (47) found that blocking PD-L1 significantly increased the severity of AHR in the HDM-exposed C3H/HeJ mice compared to the IgG-control mice. They observed that the PD-1/PD-L1 blockade enhanced pulmonary neutrophilia and IL-17A production without affecting the Th2 cytokine synthesis of IL-4, IL-5, and IgE levels in vivo. They also cultured naive T cells under Th1⁻, Th2⁻, or Th17^-polarizing conditions in the presence of IgG-Fc or rPD-L1. Similar to the Th1⁻ and Th17^– polarized cells restimulated in the presence of rPD-L1, the presence of PD-L1-Ig during polarization, decreased cytokine production of both Th1 (IFN-γ) and Th17 (IL-17A and IL-17F) cells. Conversely, the presence of rPD-L1 during Th2 polarization did not significantly affect IL-5 and IL-13 production. They observed that the PD-1/PD-L1 axis constrains the development of severe allergic asthma by targeting Th17-cell activity.

In the present study, we found that the PD-L1-blocked mice had reduced pulmonary neutrophils and mucus production compared to the NA mice and the IgG-control NA mice. Further, the levels of pulmonary neutrophils and mucus production in the PD-L1 Ab-treated mice were higher than those in the PBS control mice. The concentrations of IL-17, IL-4, G-CSF, and TNF-α in the plasma and IL-17, IL-6, and G-CSF in the BALF of the PD-L1 Ab-treated NA mice were significantly lower than those of the NA mice. The concentration of IFN-γ in the plasma and the BALF of the PD-L1 Ab-treated mice was lower than that in the PBS control mice; however, there was no significant difference between the NA mice and the IgG-control NA mice. We found that PD-L1 Ab treatment decreased pulmonary neutrophil infiltration and airway inflammation by restricting Th2 and Th17 cell responses. Conversely, the PD-L1 Ab treatment did not significantly affect the Th1 cell responses of the NA mice. PD-L1 Ab treatment has been shown to decrease neutrophil infiltration by inhibiting the function of Th2/Th17 in the NA mice, partially blocking the progression of asthma and reducing airway inflammation. However, this blocking effect is not complete. This may be due to the APCs that exist on other co-stimulatory molecules of the B7 family, such as B7-1, B7-2, B7-H2, and B7 -H3, which are also involved in T and B lymphocyte activation and immune responses. Notably, it is possible that PD-L1 Ab could be applied to ameliorate NA in patients. However, the exact molecular signaling pathways responsible for the production of these cytokines and co-stimulatory molecules remain largely unknown. Thus, we will increase our sample sizes in future studies to gain further insights into the mechanisms driving Th cell differentiation and co-stimulatory molecules to improve our understanding of NA.

This study had some limitations. First, while we analyzed the cytokines in the BALF of pediatric patients, we did not collect plasma samples; however, doing so in a future study would enable comparisons to be made. We also intend to design experiments in vitro to verify the findings of our study. Finally, our clinical cases and mice model sample size was relatively small; however, due to our desire to reduce the use of animals, we chose to limit the number of animals to 4–6 animals per group for this asthma study. Research with larger sample sizes needs to be conducted in the future to gather further evidence to support our findings.

Conclusions

In conclusion, our results suggested that PD-L1 may contribute to the Th1/Th2/Th17 immune response, which is associated with neutrophilic inflammation and asthma, and affects Th17 and its related cytokines (IL-17, IL-6, G-CSF). In NA, a PD-L1 blockade may decrease neutrophil infiltration and mucus production by inhibiting the function of Th2/Th17.

Acknowledgments

Funding: This work was supported by the following foundations: (I) The National Natural Science Foundation of China (Grant to WJ, No. 81570016); (II) The National Science Foundation of China (Grant to ZRC, No. 81771676 and 81970027); (III) The Health Department Project of Jiangsu Province, China (Grant to ZRC, No. Q201403); (IV) Science and Technology Plan Projects of Suzhou, Jiangsu Province, China (Grant to ZRC, No. SYS201350).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://dx.doi.org/10.21037/atm-21-5648

Data Sharing Statement: Available at https://dx.doi.org/10.21037/atm-21-5648

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://dx.doi.org/10.21037/atm-21-5648). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the Institutional Human Ethics Committee of the Children’s Hospital of Soochow University (No. 2014009), and written consent was obtained from the parent/guardian of each child participating in this study. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The animal experiments were performed according to the European Community Guidelines for the Care and Use of Animals and approved by the Ethics Committee for Animal Use of Soochow University (No. SUDA20200510A02).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: L. Huleatt)