Dendritic cells trigger imbalance of Th1/Th2 cells in silica dust exposure rat model via MHC-II, CD80, CD86 and IL-12

Silicosis is one of the most common occupational respiratory diseases caused by inhaling silica dust over a prolonged period of time, and the progression of silicosis is accompanied with chronic inflammation and progressive pulmonary fibrosis, in which dendritic cells (DCs), the most powerful antigen presentation cell (APC) in the immune response, play a crucial role. To investigate the role of DCs in the development of silicosis, we established an experimental silicosis rat model and examined the number of DCs and alveolar macrophages (AMs) in lung tissues using immunofluorescence over 84 days. Additionally, to obtain an overview of the immunological changes in rat lung tissues, a series of indicators including Th1/Th2 cells, IFN-γ, IL-4, MHC-II, CD80/86 and IL-12 were detected using flow cytometry and an enzyme-linked immunosorbent assay (ELISA) as well as a real-time polymerase chain reaction (PCR) assay. We observed that the number of DCs slightly increased at the inflammatory stage, and it increased significantly at the final stage of fibrosis. Polarization of Th1 cells and IFN-γ expressions were dominant during the inflammatory stage, whereas polarization of Th2 cells and IL-4 expressions were dominant during the fibrotic stage. The subsequent mechanistic study found that the expressions of MHC-II, CD80/86 and IL-12, which are the key molecules that connect DCs and Th cells, changed dynamically in the experimental silicosis rat model. The data obtained in this study indicated that the increase in DCs may contribute to polarization of Th1/Th2 cells via MHC-II, CD80/86, and IL-12 in silica dust-exposed rats.


Introduction
Silica dust is one of the most common environmental and occupational risk factors; long term exposure to silica dust contributes to the development of a number of diseases including silicosis, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). 1 Due to lack of widely accepted criteria for diagnosis or classication of autoimmunity and animal models that mimic silica dust exposure in humans, studies related to the diseases caused by silica dust have always focused on silicosis. 2,3 Silicosis is a brotic lung disease caused by the inhalation of silica dust. Occupational exposure to respirable silica particles occurs in many situations, which are oen called the dusty trades and include abrasive blasting with sand, jack hammering, drilling, mining/tunneling operations, and cutting and sawing. 4 The prevalence of disorders associated with silica dust exposure is widely observed, especially in low and middle income countries, where actual cases are oen under-reported because of poor surveillance. 5 To date, the pathogenesis of silicosis is still unclear. Numerous studies have proposed several mechanistic hypotheses, which are not systematic and remain tentative. [6][7][8][9] The immune hypothesis has been conrmed with high consensus in silicosis research. 10 Silicosis is a complex immune process including the identication, uptake and presentation of silica dust, which triggers and regulates the immune response through mechanisms that have not been established. The Si-OH complex of silica and H 2 O is similar to pathogen-associated molecular patterns (PAMPs) and therefore, it is recognized and bound by pattern recognition receptors (PRRs) on AMs; 11 it can induce AMs to secret cytokines and chemokines to initiate the inux of inammatory cells such as macrophages, neutrophils, and lymphocytes. 12,13 Upon activation of the innate immune system, silicosis presents an acute inammatory reaction at an early stage, which is characterized by inltration of inammatory cells and destruction of alveolar walls. Accompanied by the activation of inammatory cells and the secretion of cytokines and chemokines, adaptive immunity is also involved in the development of silicosis. Many broblasts are activated and proliferated, releasing a large amount of collagen; silicosis progresses to diffuse interstitial brosis or eventually form silicotic nodules. 14,15 Previous studies have demonstrated that CD4 + T cells are considered as the key participant in silicosis, and Th1/Th2 cells participate in the pathogenesis of silicosis. 16,17 DCs are the most efficient APCs that can activate both T cells and B cells and thus, they act as a bridge between innate and adaptive immunity. [18][19][20][21] Besides, DCs also have the ability to inuence T cell polarization via three ways: (i) antigen presentation, (ii) co-stimulatory molecule expression, and (iii) direct contribution by DCs to the immediate cytokine milieu that directs the resultant Th cell response. 22 MHC-II, CD80, CD86, and IL-12 have been shown as key molecules that connect DCs and Th cells in immunological diseases. [23][24][25] In addition, CD86 and IL-12 are crucial for Th1 priming, whereas no exact mechanism for the regulation of Th2 exists. 26,27 Recent studies have indicated that DCs are associated with brotic diseases. [28][29][30] Studies on human brotic interstitial lung diseases have also demonstrated that the resident cells in pulmonary brosis can sustain chronic inammation by driving the accumulation of DCs with the potential to mature locally within ectopic lymphoid follicles. 31 However, there is no relevant study on the regulation of Th1/Th2 cell polarization by dendritic cells in silicosis.
This study was designed to determine whether silicosis is associated with DCs using the rat model of experimental silicosis; it was also designed to know whether polarization of Th1/Th2 cells by DCs is involved. Herein, we characterized the number of DCs and examined Th1/Th2 cells and the expression of cytokines up to 84 days to assess potential mechanisms of silicosis.

Experimental animals
Male Sprague-Dawley (SD) rats (age: 6-8 weeks; weight: 180-220 g) were purchased from the Laboratory Animal Center of Henan Province (Zhengzhou, China). All rats were kept at the Zhengzhou University specic-pathogen-free (SPF) laboratory animal facility. Cages, bedding, and food were sterilized by autoclaving. All experimental procedures were performed in strict accordance with "Principles of Laboratory Animal Care and Use in Research" (State Council of China, 1988) and were approved by the Institutional Animal Care and Use Committee of Zhengzhou University (Zhengzhou, China).

Generation of animal silicosis model
Eighty-four rats were divided into treatment and control groups; 6 from each group were euthanized on 1 st , 7 th , 14 th , 21 th , 42 th , 63 th and 84 th day post injection. Silica (SiO 2 purity >99%, average particle size 0.5-10 mm, Sigma-Aldrich, Shanghai, CN) was ground and dried. Particulates were suspended in sterile saline at a concentration of 100 mg ml À1 . Prior to endotracheal instillation, penicillin (North China Pharmaceutical Co. Ltd., Shijiazhuang, CN) was added at 2000 units per ml. The rats were anesthetized with ether and then were hung on the metal shelf by hooking the string with their teeth. Endotracheal intubation was performed when the tracheal opening was seen from the rat's mouth. Aer successful intubation, 1 ml of SiO 2 suspension was rapidly pushed into the trachea and then, 2 ml of air was pushed into the trachea. The intubation tube was rapidly pulled out, and the suffocation was lied as soon as possible. Saline exposure rats were injected with the same volumes of sterile saline and penicillin in the same manner.

Euthanization procedure
Rats were euthanized with a sealed euthanasia device, which had good transparency and a convenient window to observe whether the animal died or not. Before the rats were placed into the device, we put a certain amount of carbon dioxide into the device, so that the rats could enter anesthesia faster with reduced fear and pain. Moreover, carbon dioxide was continuously passed for 2 to 3 minutes aer the rats were euthanatized.

Immunouorescence (IF)
The expressions of CD68 and OX-62, which are specic biomarkers of AMs and DCs, respectively, were observed in lung tissues using double-labeling immunouorescence. [32][33][34] All rats were sacriced by luxation of cervical vertebra to collect the same section of the right lung tissue, which was then xed in 10% neutral formalin and embedded in paraffin. Paraffin sections were deparaffinized, rehydrated in xylene and ethanol and then treated with 3% H 2 O 2 (Boster Biological Technology, Ltd, Wuhan, CN) for 10 min to suppress endogenous peroxidase activity and reduce background staining. Aer heating in citrate buffer (Boster Biological Technology) for 20 min, the sections were blocked with 10% goat serum (Boster Biological Technology) in TBS for 1 hour at room temperature. These sections were then incubated overnight at 4 C with mouse anti-rat OX-62 (dilution 1 : 50, BD Pharmingen, San Jose, CA, USA) and rabbit anti-rat CD68 (dilution 1 : 200, Abcam, USA) for immunouorescent double staining. Next, the sections were incubated with TRITC goat anti-mouse IgG for mouse anti-rat OX-62 (dilution 1 : 400, Boster Biological Technology) and FITC goat anti-rabbit IgG for rabbit anti-rat CD68 (dilution 1 : 400, Boster Biological Technology) and mounted under coverslips, sealed with nail polish to prevent drying and movement under the microscope. We randomly selected 5 visual elds (Â400) for each slice; positive staining for OX-62 was indicated by red staining, and CD68 positivity was indicated by green staining. The Image-Pro Plus 6.0 soware was used to analyze the number of positive cells in each photo.  and RNase-free water. This reaction mixture was incubated at 37 C for 40 min, followed by enzyme inactivation at 70 C for 15 min. cDNA samples were stored at À20 C until further processing. Real-time PCR was performed in the StepOnePlus real-

Flow cytometry
Lungs were minced and incubated in RPMI-1640 (Gibco, Grand Island, NY, USA) with 2 mg ml À1 collagenase D for 30 min in 37 C. Aer digestion, the lung cells were dispersed by gentle pipetting, followed by ltration through a 75 mm cell strainer. Then, 20 ng ml À1 of phorbol 12-myristate 13-acetate and 1 mg ml À1 of ionomycin (Sigma-Aldrich) were added to the cells. The cells were incubated at 37 C in 5% CO 2 for 1 hour and then supplemented with 10 mg ml À1 Brefeldin A (Sigma-Aldrich, Shanghai, CN) and incubated for another 4 hours. The cells were resuspended in 100 ml of staining buffer containing FITC-labeled mouse anti-rat CD3 and PerCP mouse anti-rat CD8a (BD Pharmingen) antibodies, and they were incubated for 30 minutes at 4 C, xed and permeabilized by addition of 500 ml of xation/permeabilization solution (BD Pharmingen), vortexed and then incubated at RT in the dark for another 20 minutes. Subsequently, the cells were resuspended in 100 ml of Perm/Wash buffer containing PE mouse anti-rat IL-4 and Alexa Fluor® 647 mouse anti-rat IFN-g antibodies and stained for 30 minutes at 4 C. Mouse IgG1 PE isotype control (BD Pharmingen) and mouse IgG1 Alexa Fluor® 647 isotype control (BD Pharmingen) were used as negative controls. All samples were washed, resuspended in 2% paraformaldehyde and analyzed by ow cytometry (Accuri C6, BD Accuri).

Cytokine enzyme-linked immunosorbent assay (ELISA)
A total of 100 mg of the same sections of the rat lung tissue, previously used for the immunouorescence study, was ground on ice. The tissue samples were then centrifuged, and the resulting supernatant was collected and analyzed for IFN-g and IL-4 production using murine cytokine ELISA kits according to the manufacturer's protocol (Boster Biological Technology).

Statistical analysis
All data were collected and analyzed using Microso Excel 2013 (Microso, Redmond, WA, USA) and SAS 9.2 for windows (SAS Institute Inc., Cary, NC, USA) respectively, and the values of continuous variables were expressed as mean AE standard error of mean (SEM). Differences between any two independent  samples under normal distribution were compared using Student's t-test. A P value less than 0.05 was considered as statistically signicant unless otherwise indicated.

Characterization of pathological changes of lung tissue from silicotic rats
Clear inltration of inammatory cells and a thickened alveolar septum were observed on the 1 st day and 7 th day post injection. On the 21 th day, several nodules with increased cellularity and some brotic nodules were observed among lung tissues, blood vessels and bronchus. Interstitial edema, haemorrhage and inltration of inammatory cells such as macrophages and lymphocytes were observed in interstitial nodules. Many cellular nodules, brotic nodules and fused pulmonary alveoli were observed on 42 th , 63 th and 84 th days, and considerable fusion was found among gaps of nodules and fractures of the alveolar septum (Fig. 1).

Response of AMs and DCs to silica dust exposure
We performed double-labeling immunouorescence on DCs and AMs in lung tissue sections ( Fig. 2A). The number of AMs signicantly decreased in rats from the 1 st day of silica dust exposure and reached the lowest level on the 7 th day; then, it increased and returned to the normal level on the 21 th day. The level of AMs continued to increase and reached the highest level on the 42 th day, and this level was maintained till the end of the observation. The difference in AM numbers between silica dust exposure rats and control was statistically signicant at each time point except for the results of the 21 st day (P < 0.05); the number of DCs increased from the 1 st day, and even more DCs were observed on the 42 th day in the silica dust exposure rats (P < 0.05). These data demonstrated that the number of AMs and DCs changed upon exposure to silica dust ( Fig. 2B and C).

Expression of Th1/Th2 cells and cytokines response to silica dust exposure
We performed ow cytometry analysis to detect intracellular IFN-g and IL-4 expressions in CD4 + T cells in single cell suspensions of lung tissues (Fig. 3A). The subsets of Th cells in silica dust exposure rats changed remarkably compared to those of the controls. The proportion of Th1 cells in the silica dust exposure rats increased rapidly aer exposure to silica; then, it decreased gradually with dust time. The difference between silica dust exposure rats and the control was statistically signicant on 1 st , 7 th and 14 th days (P < 0.05) (Fig. 3B). Inversely, the proportion of Th2 cells in silica dust exposure rats increased gradually with the increase in dust exposure time; except on 1 st and 7 th day, the difference between silica dust exposure rats and control was statistically signicant (P < 0.05) (Fig. 3C). We therefore performed ELISAs to detect IFN-g and IL-4 in lung tissue, and we found that Th1 cells and IFN-g as well as Th2 cells and IL-4 had similar trends. IFN-g expression increased during the inammatory stage (P < 0.05) (Fig. 4A), whereas IL-4 expression increased in the brotic stage (P < 0.05) (Fig. 4B). Based on these ndings, it was apparent that the polarization of Th1/Th2 cells exists in silica dust exposure rats, which may contribute to the development of silicosis.

Costimulatory molecules in OX-62 + DCs that regulate Th1/Th2 cells
To investigate whether DCs could regulate Th1/Th2 cells, the costimulatory molecules CD80, CD86, MHC-II and IL-12 in OX-62 + DCs were detected using real-time PCR. The results showed elevated gene expressions for MHC-II, CD80, CD86 and IL-12 in silica dust exposure rats. The difference in expression for CD80 at all time points except on 1 st day, for CD86 and MHC-II at all time points except on 84 th day, for IL-12 on 1 st , 7 th , 14 th , and 21 th day between silica dust exposure rats and control was statistically signicant (P < 0.05) (Fig. 5). These results conrmed that DCs may regulate Th1/Th2 cells by these costimulatory molecules in silica dust exposure rats; however, the determination of the molecules regulating Th1 or Th2 cells requires further study.

Discussion
Exogenous antigens enter the human respiratory system and induce inammatory and anti-inammatory responses that can result in immune dysfunction including tolerance of autoantibodies and ambient particles as well alteration of the ability to respond to exogenous pathogens and microorganisms. 35,36 Multifunctional DC cells play a central role in innate and adaptive immunity upon pathogen exposure, and they capture, process and present antigens, activate other immunocytes and eliminate debris and other materials. 37,38 In the present study, we detected the number of AMs and DCs in rat lung tissue over 84 days by ow cytometry. The results demonstrated that the number of AMs signicantly decreased, whereas DCs showed the opposite trend during the inammation period of silicosis. These results were in accordance with the results reported by Beamer, who demonstrated that the percent and absolute number of AMs decrease signicantly with concomitant signicant increase in DCs. 39 This suggested that compared with AMs, DCs are more tolerant when attacked by silica dust and may have primary role in identication, uptake and presentation of silica dust to activate T lymphocytes. Subsequently, during brosis, AMs and DCs both increased signicantly, participating in brosis to create and sustain a probrotic lung microenvironment 40,41 . In addition, our observation time was much longer than that of Beamer, and a longer period was benecial for determining the complete trends of AMs and DCs. Polarization of Th1/Th2 cells and cytokines has been conrmed in immunity-related diseases, 42,43 and many studies have conrmed that it also plays an important role in the process of brosis. [44][45][46] To date, most silicosis models have only detected Th1/Th2 cells and cytokines within 28 days. 47 However, the occurrence and development of silicosis is a gradual and prolonged process, and the entire process of silicosis cannot be reected in 28 days. Therefore, we dynamically examined Th1/ Th2 cells and IFN-g and IL-4 cytokines for up to 84 days. The results showed that compared with the observations for the control, the polarization of Th1 cells and IFN-g expressions signicantly increased during the inammatory stage in silica exposure dust rats and that the polarization of Th2 cells and IL-4 expressions signicantly increased during the brotic stage of silicosis. It was demonstrated that the Th1 cells were predominant and activated in the inammation period of silicosis and secreted large amounts of IFN-g to inhibit brosis; in brosis during silicosis, the Th2 cells were predominant and activated, and they secreted IL-4 to promote pulmonary brosis. Our ndings are consistent with Wu's study of INF-g dominance (Th1 cytokine) in pigeon breeder's lung patients at the acute/ sub-acute stage and IL-4 dominance (Th2 cytokine) at the chronic stage aer pulmonary brosis occurred. 48 Recent studies have found that DCs can regulate polarization of Th1/Th2 cells in a variety of diseases, but this regulation in silicosis is rarely reported. [49][50][51] Then, we detected CD80, CD86, MHC-II and IL-12 expressions to conrm whether these molecules produced by DCs can initiate Th cell polarization in silica dust exposure rats. The results suggested that DCs presented silica dust to T cells via elevated expressions of CD80, CD86, MHC-II and IL-12 and thus regulated the polarization of Th1/ Th2 cells in silica dust exposure rats. However, the determination of the specic cytokine that regulates Th1 or Th2 cells during such exposure still needs to be researched.
In summary, our study demonstrated that DCs accumulated in lung tissues of silica dust exposure rats and regulated the polarization of Th1/Th2 cells via CD80, CD86, MHC-II and IL-12 expressions, indicating that DCs may play a critical role in modulating immune homeostasis during silicosis in rats. However, the detailed mechanism of DCs regulating the polarization of Th1/Th2 cells remains to be further investigated.

Author contributions
CF and WY designed the study. LB, SN, JW, and LZ conducted experiments, analyzed the data, and draed the manuscript. All authors read and approved the nal manuscript.

Conflicts of interest
None of the authors has a nancial relationship with a commercial entity that has an interest in the subject of this manuscript.