Effect of hydrogen inhalation on IL-40 and SIgA in a Rat Model of Pulmonary Mucosal Immunity

Background Recently, some researchers have reported that PIgR expression is down-regulated in Chronic Obstructive Pulmonary Disease (COPD) and SIgA deficiency correlates with severity of airflow obstruction. What’ s more, some studies have demonstrated that 2 percent of hydrogen or hydrogen water is effective in treating and preventing various diseases. Objectives The aim of this study was to observe the effect of hydrogen on the expression of SIgA, PIgR, IL-4, IL-5, TGF-β1 and IL-40 in lung tissue of COPD rats, to study the relationship between lung pathology parameter and SIgA, PIgR, therefore we can understand the effect of hydrogen on the development of COPD by changing SIgA expression of airway mucosal in COPD rats. Methods A rat model of COPD was established by cigarette smoke exposure, and different concentrations of hydrogen were inhaled as intervention measures. After 4 months of cigarette smoke exposure, pathologic changes and airway wall remodeling of the lung were assessed by optical microscope. The protein expressions of SIgA, PIgR, IL-4, IL-5, TGF-β1 as well as IL-40 in the lung tissues were observed by immunohistochemistry or Western blot. The correlation between lung pathology parameter and the expression of SIgA, PIgR was analyzed. The correlation between SIgA and the expression of IL-4, IL-5, TGF-β1 and IL-40 was analyzed. Results The results showed that hydrogen inhalation significantly ameliorated lung pathology and airway wall remodeling, increased the protein expression of SIgA, PIgR, IL-4, IL-5, and IL-40, and reduced the protein expression of TGF-β1. Conclusions Inhalation of 22% and 41.6% hydrogen showed a better effect than inhalation of 2% hydrogen. Hydrogen inhalation can significantly improve the expression of SIgA on the mucosal surface of COPD rats, which may be one of the mechanisms which hydrogen works on COPD pathogenesis.


Introduction
Chronic Obstructive Pulmonary Disease (COPD) is a common, preventable and treatable disease, which is characterized by irreversible airflow limitation due to airway and/or alveolar abnormalities, usually caused by significant exposure to harmful particles or gases (1,2). It is currently reported to be the fourth leading cause of death worldwide, and also expected to be the third leading cause of death by 2020 (3). Recently, some researchers have found that mucosal deficiency is related to chronic airway inflammation. Subsequently, the study has shown that local IgA deficiency is associated with bacterial translocation, small airway inflammation, and airway remodeling (4).
Surface IgA (SIgA), along with mucociliary clearance, defenses against inhaled antigens and microorganisms by preventing airway inflammation (5). In small airways, IgA is produced locally by subepithelial plasma cells. A dimer of two IgA monomers linked by J("joining") chain and secretory component (SC) composes SIgA (6), which is transported across airway epithelial cells via the polymeric immunoglobulin receptor (pIgR). After transportion to the mucosal surface, pIgR is cleaved and SC remains attached to form SIgA (7). SC is an extracellular component of pIgR, which plays a role in stabilizing the structure of SIgA in the process of transportion (8). Therefore, SIgA and SC play a pivotal role in the immunity of respiratory tract (9,10). In 2001, Pilette et al reported that SC expression was reduced in airways of COPD (11). Subsequently, researchers found that patients with COPD had reduced SIgA in bronchoalveolar lavage (12). Polosukhin et al have shown that pIgR expression is down-regulated in COPD and that SIgA deficiency is correlated with severity of airflow obstruction(13).
Richmond et al have documented that, as a consequence, pIgR deficiency leads to chronic neutrophil inflammation, which contributes to small airway fibrosis and emphysema (14). Together with what has been published above , dysfunction of bronchial epithelial mucosa led to abnormalities in SIgA transported to the airway, which impairs the defense ability of local hosts. Therefore, persistent structural and functional abnormalities of airway epithelium play a key role in development and progression of COPD.
Cytokine, acts as a secreted protein, may plays a vital role in host defense, inflammation, and immune responses. In 2017, Catalan et al described the latest cytokine, IL-40, which was found to be expressed by peripheral B cells upon activation, and encoded a small secreted protein(27kDa) of 265 amino acids by an uncharacterized gene (15).
What' s more, they also found IL-40-/-mice exhibited smaller Peyer' s patches, lower numbers of IgA-secreting cells, and fewer IgA in the serum, gut, feces, and lactating mammary gland (16). Interestingly, TGF-β1 had been shown to play an important role in potentiating the expression of IL-40 by activated B cells, and a progressive increase was observed when B cells were treated with IL-4 and TGF-β1. There was a direct correlation between the expression of IL-40 and the onset of IgA production. Understandably, IL-40 was involved in the development of humoral immune responses, especially those associated with IgA production.
Together, our researches shed light on the potential mechanism of SIgA expression in COPD rats. In recent years, a number of studies have shown that hydrogen therapy has a positive effect on various diseases (17)(18)(19)(20). Liu et al in our team found that different concentrations of hydrogen inhalation could significantly reduce inflammation and apoptosis, protect mitochondria, and ameliorate cardiovascular function in COPD rat model (21). A key question is whether there exist a possibility that hydrogen inhalation will have a positive effect on SIgA expression in rat model of COPD , which is to be tested in the experiment. An animal model was established by the smoking method. All animals were randomly divided into 5 groups (10 rats in each group):

Instruments and reagents
control group, COPD group, high-hydrogen group (Hh group, which was exposed to 21% O 2 , 41.6% H 2 , and 100% N 2 at a flow rate of 5:3), intermediate hydrogen group (Hm group, which was exposed to 21% O 2 , 22% H 2 , and 15% O 2 , 85% N 2 at a flow rate of 1:2), and low-hydrogen group(Hl group, which was exposed to 21% O 2 , 2% H 2 ). In addition to the control group, cigarette smoke and hydrogen were in reality exposed for 4 months. In the meantime, the control group received no intervention during 4-month period.

Tissue Preparation
Tissue preparation was performed as described previously (22). The lung was harvested and fixed immediately in buffered 4% paraformaldehyde for 24 h. The tissue was subsequently dehydrated in a graded ethanol series and embedded into paraffin. For histomorphology analysis,

Masson's trichrome staining
Masson's trichrome staining was performed by standard protocol. The value of bronchial airway was selected which was defined as the difference between the area delineated by the inner boundary of tunica intima (A1) and the external boundary of tunica adventitia (A2), divided by the length of the external boundary of tunica intima. The area of the wall (A3) was calculated with A1 and A2 (A3 = A2 -A1), and the ratio of wall and lumina was achieved with A3 and A2 (the ratio = A3/A2).
The bronchial wall thickness/ diameter (BWT/D) ratio was determined.

IL-5 in Pulmonary Tissue
The protein expression levels of SIgA, IL-4 and IL-5 were assessed by IHC. Before using microwave antigen retrieval, deparaffinized sections were pretreated, followed by incubation in 3% H 2 O 2 at 25℃ for 25 min, and goat serum at 37℃ for 40 min. Next, the tissues were incubated respectively with primary antibodies overnight at 4℃. After rinsed with PBS, the tissues wereincubated with biotinylated secondary antibody at

Statistical Analysis
Data were presented as the mean±standard deviation (SD).

The pathological changes of lung tissues in COPD rats with hydrogen inhalation
In control group, the alveolar structure was normal, the trachea mucosal was intact, pulmonary artery wall structure was normal, and no inflammatory cell infiltration was found. In COPD group, alveolar duct, alveolar vesicle and alveoli were significantly enlarged, alveolar structure was disordered, alveolar wall was broken, pulmonary bullae was partial fusion, ciliated cell were degeneration and necrosis, the airway wall thickness increased obviously, the airway mucosal was damaged seriously, and the airway wall remodeling increased obviously, pulmonary arteriole wall was thickening, and different levels of infiltration by neutrophils, lymphocytes and monocytes were observed.
Compared with COPD group, the thickening of alveolar wall, airwway wall and pulmonary artery wall was ameliorated, the condition of alveolar fracture was better, inflammatory cell infiltration and the number of bullae was less in Hh, Hm and Hl groups (Fig 1A).  (Fig 1B).

The expression of SIgA, IL-4 and IL-5 in lung tissue was detected by immunohistochemistry
Compared with control group, the expression of IL-4, IL-5 was reduced in COPD group. In contrast with COPD group, IL-4, IL-5 was increased in Hh group and Hm group. Compared with COPD group, IL-4, IL-5 show no difference in Hl group (Fig 3A). Compared with control group, SIgA was significantly reduced in COPD group (P<0.01). Compared with COPD group, SIgA was significantly increased in Hh group and Hm group (P<0.01), there was no significant difference in Hl group (P>0.05), but there was no significant difference between Hh group and Hm group (P>0.05) (Fig 2A, 2B).  (Fig 2A, 2B, 3B, 3C).

Discussion
Bronchial epithelial SIgA is the critical antibody for respiratory immune defense against inhaled antigens and microorganisms at mucosal surfaces, including the airways. Recently, studies have shown that SIgA deficiency is widespread in COPD. Therefore, how to improve the bronchial mucosal immunity is a core issue. Our previous study showed that hydrogen inhalation could visibly reduce inflammation and apoptosis, and improve cardiovascular function, suggesting a "significant effect" of hydrogen inhalation on COPD (21). Our results provided new information that treatment with hydrogen could ameliorate pathological changes, airway remodeling as well as SIgA expression. Moreover, we found that Hm hydrogen concentration was necessary for SIgA expression. Together, our findings pinpointed a positive role of hydrogen inhalation in improving mucosal immunity in development and progression of COPD.
In this study, COPD model was prepared by cigarette smoke exposure method. The experimental results showed that the animal model of COPD rats was made successfully from the behavioral activity and lung pathological changes. In lung pathological changes, the effect of low concentration of hydrogen is poor, the mechanism may be the antioxidant effect of hydrogen, low concentration of hydrogen can not resist the activation of the body to produce too much oxygen free radicals, and inhaled high concentration of hydrogen just neutralize this reaction. The mechanism may be the anti-inflammatory effect of hydrogen, which can reduce the release of inflammatory mediators and decrease the inflammatory factors to improve lung pathological status in COPD rats.
The experiment found that SIgA and PIgR decreased in COPD group, which may be due to the continuous stimulation of harmful gases, resulting in the destruction of bronchial mucosal structure, and decreased in the number of plasma cell, decreased IgA secretion (23) and reduced carrier PIgR. On the one hand, the composition and SC of synthetic carriers were reduced, on the other hand, the carrier was reduced, and finally the SIgA transported to the bronchial mucosal surface was reduced.
Inhalation of high-concentration hydrogen significantly increased SIgA expression, and the effect was better than that of low-concentration hydrogen. This may be that the medium-high concentration of hydrogen stimulates plasma cell production, resulting in increased IgA secretion and PIgR delivery, so the SC of raw materials for synthetic SIgA will also increase, thus ameliorating the lack of airway mucosal surface.
The experimental study found that the expression of IL-4 was significantly decreased in COPD group, which may be caused by smoke stimulation Th cell dominant differentiation (24,25). The expression of IL-4 and SIgAwas increased in Hh and Hm group, and the two closely related, so IL-4 may be one of the important factors affecting the expression of SIgA on the airway mucosal surface of rats with enhanced hydrogen. The hydrogen treatment group increased IL-5 expression to varying degrees. We hypothesized that hydrogen might increase IL-5 production by affecting Th2 cells, IL-5 promoting mucosal IgA production (26). A deficiency of the experiment is the lack of detection of Th2 cells, so whether the above views are true or not needs to be further improved to confirm. We speculate that the other possible mechanism SIgA enhancement is that hydrogen can activate peripheral B cells. IL-40 participates in humoral immune response, and affects the number of IgA secretory cells, resulting in an increase in IgA production (16). IL-40, however, as a newly discovered factor in the last two years, few studies have been conducted to investigate whether IL-40 greatly affects SIgA expression as well as lung pathology. TGF-β family plays an important role in the development of human tissues and homeostasis. It regulates multiple cellular processes and is closely related to tissue remodeling in pulmonary fibrosis and emphysema (27). A number of studies have shown that TGF-β1 antagonists can ameliorate lung function, reduce collagen deposition, ameliorate lung pathology and airway remodeling (28,29).
The results of this experiment show that hydrogen can effectively reduce the expression of TGF-β1, and statistics show that the expression of TGF-β1 is negatively correlated with the expression of SIgA. We believe that hydrogen interferes with lung pathology by down-regulating the expression level of SIgA.

Conclusions
The model of COPD rats was successfully established after 4 months of smoke exposure. Through pathology observation and index measurement, hydrogen inhalation can ameliorate lung pathology injury in COPD rats.