Effects of Overexpressed SOCS1 Transfected DCs on Th17- and Treg-Related Cytokines in Mice with COPD

Background: In this study, we established a chronic obstructive pulmonary disease (COPD) model by stimulating mice with cigarette smoke, and observed the effects of dendritic cells (DCs) overexpressing SOCS1 on Th17, Treg and other related cytokines in peripheral blood, bronchoalveolar lavage uid and lung tissues of COPD mice. Methods: After successfully transfecting DCs with overexpressing SOCS1 (DC-SOCS1), the mice were injected with DC-SOCS1 (1×10 6 ), DC-SOCS1 (2×10 6 ) and immature DCs (1×10 6 ) via tail vein on days 1 and 7 of COPD fumigation modeling. After day 28 of modeling, the peripheral blood, BALF and lung tissue samples were extracted from the mice, and the changes of DCs, Th17 and Treg cells and related cytokines were detected by immunohistochemistry, immunouorescence, HE staining, ow cytometry and ELISA. Results: The results showed that DC-SOCS1 was able to reduce the secretion of pro-inammatory factors and increase the anti-inammatory factors in the COPD mice, and the effect of high concentration (2×10 6 DC-SOCS1) was better than low concentration (1×10 6 DC-SOCS1). Moreover, the intervention effect was signicant on day 1 compared with day 7. In the mice injected with DC-SOCS1, the expression of CD83, IL-4, Foxp3, and CCR6 was increased on day 1 than those on day 7, while IL-17 and IFN-γ was decreased. Conclusions: Intervention of COPD mice with high concentrations of DCs-SOCS1 reduced pro-inammatory factor secretion and attenuated the inammatory response in COPD. Trial registration: Not applicable.

appropriate concentration with Staining Buffer: 2-0.03 μg/10 6 cells) were added to each ow assay tube; 50 μl Staining Buffer was added to the blank or isotype control tube. In each tube, 50 μl cell suspension (approximately 10 6 cells) was added and gently mixed. After incubation for 30 min in an ice bath, the cells were incubated with Staining Buffer (2 ml per tube and 200 μl per well) and centrifuged at 1000 rpm for 5 min at 4 ℃. The supernatant was discarded and washed three times. 100 μl of the cells were resuspended and detected by FCM.

Electron microscopy
After the cells were xed, dehydrated and dried, the samples were placed on a sample stage approximately 10-15 cm away from the evaporation source and subjected to conductive treatment to observe the morphology of the induced mDCs under electron microscopy.

Construction of SOCS1 gene overexpression lentiviral vector
The SOCS1 gene (Gene ID: 12703) was searched on the NCBI website, and NM_001271603.1 transcript was chosen for sequence synthesis. The Kozak sequence and EcoRI digested site were added at the 5' end, and the BamHI site was added at the 3' end. The pLVX-IRES-ZsGreen1 vector (Figure 2a) was digested with EcoRI and BamHI. The puri ed synthetic product was ligated to the linearized vector, and the ligated product was transformed into bacterial receptor cells. The grown clones were rst identi ed by enzymatic cleavage to demonstrate that the target gene had been targeted into the target vector. After sequencing and analyzing the positive clones, the constructed lentiviral overexpression plasmid vector was subjected to high-purity endotoxin-free extraction and then co-transfected with the lentiviral packaging vector in 293T cells. The supernatant was collected, puri ed and concentrated to be the high titer pLVX-SOCS1-IRES-ZsGreen1 lentivirus. The virus titer was detected by multiplicative dilution method after infection of 293T cells. The lentiviral packaging vector is a four-plasmid system consisting of pLVX-IRES-ZsGreen1, PG-p1-VSVG, PG-P2-REV, and PG-P3-RRE (Figure 2b, c, d). Of these, pLVX-IRES-ZsGreen1 is able to express green uorescent protein (GFP), and PG-p1-VSVG, PG-P2-REV, and PG-P3-RRE contain components essential for viral packaging. The results of pLVX-SOCS1-IRES-ZsGreen and negative control lentivirus titer assay were presented in Figure 2e.

Lentiviral infection of DCs
The primary DCs were isolated and cultured, and lentiviral infection was performed on the fth day after isolation. The virus solution was aspirated and added to the cells, along with 5 μg/ml of polybrene co-transfection reagent to improve the infection e ciency. Then, the 24-well plates were incubated at 37 ℃ and the cell status was observed after 8-12 hours (h). After 24 hours, the medium was replaced with fresh medium and observed under uorescence microscope after 48 hours of infection. DCs with green uorescence were those infected with lentivirus overexpressing on SOCS1 gene. Meanwhile, samples were collected for further study.
Quantitative Real-time PCR (qRT-PCR) qRT-PCR for detecting transfection effects after lentivirus infection of DCs. SYBR Green I is a uorescent dye that binds to double-stranded DNA and emits light, allowing detection of the amount of double-stranded DNA present in the PCR system based on the uorescent signal. The RNA was rst reverse transcribed into cDNA using random primers, and then speci c primers and SYBR Green I uorescent dye were designed for uorescent quantitative PCR detection. The primer sequences were: SOCS1 (ID: 12703), mSOCS1F: CGCCTGCGGCTTCTATTG, mSOCS1R: CCCGAAGCCATCTTCACG, m actin f: GAGACCTTCAACACCCCAGC, m actin r. ATGTCACGCACGATTTCCC.

Western Blot
WB was used to detect the transfection effect after lentivirus infection of DCs. The cultured DCs infected with lentivirus were lysed with RIPA lysis buffer, centrifuged at 12000 rpm, 4 ºC for 15 min, and the supernatant was collected. Then the protein concentration was determined by BCA protein assay kit. The protein was separated on 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequently transferred to polyvinylidene uoride membranes (Bio-Rad), which were subsequently blocked with Tris Buffered saline Tween (TBST) solution containing 5 % skim milk at room temperature for 2 h. The corresponding primary antibody (SOCS1: Abcam, anti-rabbit) was diluted to a certain concentration (1:500) with the blocking solution, and the nal concentration of the internal reference primary antibody was 1:1000, and then incubated overnight at 4°C. After 3 washes with TBST, they were incubated with secondary antibodies (Sigma, goat-anti-rabbit, 1:1000) at room temperature for 1 h. Finally, samples were incubated with the ECL-enhanced chemiluminescence detection kit (PIERCE) and OD values were calculated with ImageJ pro plus.
COPD modeling in C57BL/6 mice and DCs reinfusion Thirty-seven 8-week-old male SPF-grade C57BL/6 mice, weighing 20-25 g (Chongqing Tengxin Biotechnology Co., Ltd.) were obtained. The 35 mice were randomly assigned into 7 groups (B-H groups), and the 7 groups were housed in identical environments ( Table 1). The animals in each group were placed in a homemade smokebox (45cm×30cm×30cm) and smoked passively 3 cigarettes/times, 1h/times, 4 times/d (Figure 3a, b, c). The time points were 9:30, 11:00, 14:00, 15:30, 7 d, and 28 d of smoking. The general condition of each group was observed, and the body mass of each group was weighed and recorded weekly before and after fumigation to dynamically observe the effect of fumigation on the body mass of mice. After 28 d of modeling, the peripheral blood was collected from the eyeballs for FCM assay. The trachea was exposed by cutting the skin in the middle of the neck, and the trachea and alveoli were irrigated with saline (0.6 ml/time, for 3 times). The BALF was collected and centrifuged (1500r/min, 10min, 4℃), and the supernatant was used for enzyme linked immunosorbent (ELISA) assay and the BALF precipitate was taken for FCM assay. The abdominal cavity was opened, and fresh liver, spleen and lung tissues were washed in cold PBS, and part of the lung tissues were taken for FCM assay, while the rest of the tissues were stored at -80℃ for ELISA assay. The tissues were xed in 4% paraformaldehyde for more than 24h for immunohistochemistry, immuno uorescence and hematoxylin-eosin staining.

hematoxylin-eosin (HE) staining
Tissues xed in 4% paraformaldehyde xative for 24h were para n embedded, sectioned, stained with hematoxylin stain and eosin stain, and sealed with neutral resin and observed under the microscope. The nucleus became blue, while the cytoplasm became red or pink.

Immunohistochemistry (IHC)
Tissues xed in 4% paraformaldehyde xative for 24h were subjected to IHC experiments after para n embedding, sectioning, and antigen thermal repair, and the changes of IL-17, IL-4, CD83, CCR6, Foxp3 and INF-γ factor levels were detected in each group. The tissues were washed with PBS and then closed with goat serum at room temperature for 1 h. Samples were then incubated with primary antibodies overnight at 4°C (IL-17A: dilution ratio 1:200 For Treg, the 100 µl cell suspension was added to each tube, with a cell count of approximately 1x106 cells. The surface antigens were labeled according to the cell surface antigen staining method, and the appropriate amount of CD4 and CD25 antibodies were added according to the instructions and incubated for 30 min at room temperature without light. Then, the cells were washed with pre-chilled PBS and the supernatant was discarded after centrifugation. Then the cells were resuspended by adding 1 ml of Fixation/Permeabilization solution and incubated for 30 min at room temperature under dark conditions. Subsequently, 1 ml Permeabilization Buffer (diluted 1:9 in deionized water) was added, and supernatant was discarded after centrifugation. Cells were resuspended with 100ul Permeabilization Buffer, added IL-17A (for Th17)and Foxp3 (for Treg) antibody, and incubated for 30 min at room temperature without light. Finally, it was washed with ow staining buffer, centrifuged at 300 g for 5 min, resuspended to 500ul with ow staining buffer, and detect on the machine.
For DCs, the 100 µl cell suspension was added to each tube, with a cell count of approximately 1x106 cells. The surface antigens were labeled according to the cell surface antigen staining method, and the appropriate amount of CD80 and CD83 (CCR5 and CCR6 for imDCs) antibodies were added according to the instructions and incubated for 30 min at room temperature without light. The cells were washed with pre-chilled PBS and the supernatant was discarded after centrifugation. Then, the cells were washed with ow staining buffer, centrifuged at 300 g for 5 min, resuspended to 500ul with ow staining buffer, and detect on the machine.
ELISA detection of Th17, Tregs, Th1 and Th2 The ELISA kit was used to determine the levels of interleukin (

Statistical Analysis
Statistical analysis was performed using SPSS version 26.0, and the quantitative data were analyzed by one way ANOVA and shown as mean ± standard deviation. P < 0.05 was considered as statistically signi cant.

Cell culture and identi cation of DCs
The growth status of DCs in culture for 1d, 48h, after uid change, 5d, 7d and after LPS induction was shown in Figure 1a. Identi cation of DCs monolabeled with CD80 and CD83 antibodies was performed by FCM, which showed a signi cantly higher number of DCs and higher uorescence signal intensity compared with the blank control, suggesting that DCs were successfully cultured ( Figure 1b). In addition, induced mDCs were also observed under electron microscopy ( Figure 1c).

Transfection Effect Results Of Lentivirus Infection Of Dcs
The states of DCs transfected with overexpressed SOCS1 gene (DCs-Ad-SOCS1) and with GFP lentivirus no-load for 5 days (DCs-Ad-GFP) was observed under the uorescence microscope as shown in Figure 2f, with green uorescence was the target cells. In addition, the amount of double-stranded DNA present in the PCR system could be detected based on the uorescence signal intensity of SYBR Green I. The qPCR results showed that the amount of DNA in the DCs-Ad-SOCS1 group was signi cantly higher compared with the DCs-Ad-GFP and imDCs groups (control group) ( Figure 2g). WB showed that the expression of SOCS1 was higher in the DCs-Ad-SOCS1 group, indicating that the SOCS1 gene had been successfully transfected into DCs (Figure 2h, i).

Successful construction of COPD model and changes of DCs in lung tissue
The weekly changes in body mass of mice in each group before and after fumigation were presented in Figure 3d. The B group, saline reinfusion group, showed a continuous decrease in body mass after fumigation. Groups C, D and E were treated with DC-SOCS1 or imDCs reinfusion on 1d of fumigation, and mice in all three groups had a continuous increase in body mass after fumigation. Groups F, G and H received DC-SOCS1 or imDCs reinfusion on the 7th day of fumigation, and the body mass of mice in the three groups decreased at 1 week after fumigation but continued to increase from the 2nd week, which meant that the body mass decreased before receiving the reinfusion and increased after the reinfusion treatment. That was to say, the body mass of mice in D and G groups that received DC-SOSC1 2×10 6 reinfusion was greater than that of C and F groups that received DC-SOCS1 1×10 6 reinfusion. Similarly, the body mass increase of mice in E group, which received imDCs reinfusion 1 d after fumigation, was greater than that in H group, which received imDCs reinfusion 7 d after fumigation. In addition, by immuno uorescence observation of lung tissue DCs, we found that compared with group A, there was a signi cant increase in DCs in group B, E and H, more growth in group C and F, a slight increase in group G and no signi cant change in group D (Figure 3e).

Content of mDCs
In the peripheral blood of mice, the content of mDCs in B group (14.25±0.50) was signi cantly higher than that in D (3.29±0.21) and E (11.37±0.15) groups (P < 0.05); the mDCs in E group was higher than that in D, F (8.98±0. 19) and G (5.13±0.28) groups with statistically signi cant differences (P < 0.05); the mDCs of F group was signi cantly higher than that of D and G groups (P < 0.05). In the BALF sediment, the content of mDCs in D group (1.98±0.11) was lower than that of E (3.58±0.09), F (3.32±0.11) and H (4.52±0.16) groups with signi cant difference (P < 0.05); the content of mDCs of G group (2.66±0.16) was signi cantly decreased compared with E and H groups (P < 0.05). However, no signi cant differences were found in the content of mDCs in Lung tissue between the groups (Figure 4a).

Content of imDCs
The content of imDCs in the peripheral blood of D group (4.46±0.23) was signi cantly higher than that of B (1.27±0.09), E (2.28±0.99) and F (2.44±1.11) groups (P < 0.05). There was no difference in the content of imDCs in the peripheral blood among the other groups. In the BALF, the content of imDCs of D group (12.92±0.01) was increased compared with B (4.92±0.18), E (5.95±0.03) and F (6.22±0.16) groups (P < 0.05); compared with B group, the content of imDCs of F group was signi cantly decreased (P < 0.05). No difference was observed in the content of imDCs in BALF among the other groups. In addition, the content of imDCs in lung tissue of F group (1.24±0.01) was higher than that of B (0.58±0.00) and H (0.64±0.01) groups with statistically signi cant differences (P < 0.05), and no differences were found in imDCs in the lung tissue among the other groups ( Figure 4b).

Content of Th17
In the peripheral blood of mice, the content of Th17 of F group (1.73±0.15) was lower compared with B (2.16±0.04), C (2.05±0.03), D (2.05±0.09) and H (2.07±0.29) groups (P < 0.05), and the content of Th17 of G group was lower than that of B, C, D and H groups (P < 0.05). Compared with B group (1.08±0.01) in lung tissue, the content of Th17 of D (0.11±0.01), G (0.28±0.03) and H (0.89±0.01) groups was signi cantly lower (P < 0.05). In addition, the content of H group in lung tissue was signi cantly higher than that of D and G groups (P < 0.05). No signi cant differences were observed in Th17 in the BALF between the groups (Figure 4c).

Content of Treg
The   Note: Data was expressed as mean ± standard deviation. Compared with group A (Normal group), + P < 0.05; compared with group B (NS group), * P < 0.05; co with group C (DC-SOCS1 I group), @ P < 0.05; compared with group D (DC-SOCS1 II group), # P < 0.05; compared with group E (imDCs I group), $ P < 0.05; comp group F (DC-SOCS1 III group), % P < 0.05; compared with group G (DC-SOCS1 IV group), & P < 0.05. ; the concentration of IL-17 in E, F and H group was signi cantly higher than that of D group (P < 0.05); the concentration of IL-17 in E and F group was signi cantly lower than in H group, but higher than in G group (P < 0.05); the concentration of IL-17 in G group was lower than in H group with statistically signi cance (P < 0.05) (Figure 5a). in C, E and F groups were increased than that of G group with statistically signi cance (P < 0.05); the concentration of IL-21 in C group was signi cantly higher than in D group but lower than in E group (P < 0.05) (Figure 5b). in C group was signi cantly higher than that of E, F, H groups but lower than that of G group (P < 0.05); the concentration of IL-4 in G group was signi cantly higher than that of E, F, H groups (P < 0.05); the concentration of IL-4 in E, H groups were signi cantly lower than in F group (P < 0.05) (Figure 5h). .08) (P < 0.05); the concentration of IL-5 in B group was signi cantly decreased than that of C, D, E, F, G groups (P < 0.05); the concentration of IL-5 in H group was signi cantly lower compared with C, D, E, F, G groups (P < 0.05); the concentration of IL-5 in E group was signi cantly decreased than that of C, D, F groups (P < 0.05); the concentration of IL-5 in C, F groups were signi cantly lower than that of D group (P < 0.05) (Figure 5i).

In lung tissue
The results showed that the concentration of IL-17 in groups C-H (13.05±0.46, 6.50±0.53, 28.12±0.33, 16.13±0.80, 8.49±0.12, 37.58±0.17) were signi cantly lower compared with B group (52.24±1.16) (P < 0.05); the concentration of IL-17 in C group was signi cantly higher than that of D, G groups but lower than that of E, H groups (P < 0.05); the concentration of IL-17 in D group was signi cantly lower than in E, F, H groups (P < 0.05); the concentration of IL-17 in E group was lower than in H group with statistically signi cance (P < 0.05); the concentration of IL-17 in E, H groups were signi cantly higher than that of F, G groups (P < 0.05) (Figure 5a). .04) were signi cantly increased than in B group (23.49±0.09) (P < 0.05); the concentration of TGF-β in C group was signi cantly higher than that of E, H groups but lower than that of D, G groups (P < 0.05); the concentration of TGF-β in D group was signi cantly higher compared with E, F, G, H groups (P < 0.05); the concentration of TGF-β in E, F, G groups were signi cantly increased compared with H group (P < 0.05); the concentration of TGF-β in G group was signi cantly higher than that of E, F groups (P < 0.05); the concentration of TGF-β in E group was lower than that of F group with statistically signi cance (P < 0.05) (Figure 5e). concentration of IFN-γ in D group was signi cantly lower than that of E group (P < 0.05); the concentration of IFN-γ in F group was signi cantly higher than that of G group (P < 0.05) (Figure 5f).
The concentration of IL-12 in groups B-G groups (40.06±0.05, 30.74±0.06, 26.07±0.18, 35.13±0.04, 32.63±0.09, 28.67±0.12) were signi cantly higher than that of A group (19.43±0.03) (P < 0.05); the concentration of IL-12 in C-G groups were signi cantly lower than that of B group (P < 0.05); the concentration of IL-12 in C, E, F, G groups were signi cantly higher than in D group (P < 0.05); the concentration of IL-12 in C, F, G groups were signi cantly decreased in comparison with group E group (P < 0.05); the concentration of IL-12 in F group was higher than that of C, G groups with statistically signi cance (P < 0.05); the concentration of IL-12 in C group was signi cantly higher compared with G group (P < 0.05) (Figure 5g). signi cantly lower compared with A group (326.85±0.81) (P < 0.05); the concentration of IL-4 in C-G groups were signi cantly increased than that of B group (P < 0.05); the concentration of IL-4 in H groups were signi cantly lower than that of C, D, F, G groups (P < 0.05); the concentration of IL-4 in D group was signi cantly higher than that of E, F groups but lower than that of G group (P < 0.05); the concentration of IL-4 in E group was signi cantly lower than that of F, G groups (P < 0.05); the concentration of IL-4 in F group was signi cantly lower compared with G group (P < 0.05) (Figure 5h).

Morphological Differences Of Copd Models
As shown in the gure, the pathological changes were signi cantly aggravated in the B, C, D, E, F, G groups compared with A group. There were signi cant changes in the C, D, E, F, G groups compared with group B, with signi cant reduction in pathological changes in groups C, D, F and G ( Figure 6).

Expression Of Dcs, Th17 And Tregs In Lung Tissue
In lung tissues of mice, the expression level of IL-17A, CCR6 and Foxp3 in F, G and H groups was increased, but the expression of CD83, IL-4 and IFN-γ was decreased compared with C, D and E groups (Figure 7).

Discussion
COPD is a chronic in ammatory disease of the airways that seriously endangers human health, characterized by a long course, di cult to cure and easy to recur, and characterized by progressive, incomplete and reversible air ow limitation (13). At present, smoking is considered to be one of the most important pathogenic factors in COPD. Smoking causes chronic in ammation in the airways, and it has been found that 80%-90% of COPD patients are smokers (14).
Chronic in ammation of the airways in COPD patients who had previously smoked was found to persist after fumigation cessation (15).
In this study, we performed smoke stimulation and injected DCs with overexpressed SOCS1 and imDCs through tail vein to observe the lung histopathological manifestations and the changes of in ammatory factors in peripheral blood, BALF and lung tissues in COPD model mice after 4 weeks. The results showed that the lung histopathological manifestations of the mice after 4 weeks of modeling were consistent with those reported in the early literature(16), and were in accordance with the typical pathological features of COPD lung tissues, and the pathological changes in the lung tissues of the mice in the DC-SOCS1 group were signi cantly reduced compared with those in the control group.
In the present study, the expression of IL-4, Foxp3, and CCR6 was increased in lung tissues of mice reinfused with DC-SOCS1 on day 1 after fumigation compared with mice reinfused with DC-SOCS1 on day 7, but the expression of CD83, IL-17 and IFN-γ was decreased. CD83 is a speci c marker of mDCs, and the amount of its expression represents the degree of DC in ltration in tissues (17). It has been proved that cigarette extracts and nicotine can affect the maturation and function of DCs, and long-term stimulation of DCs with cigarette extracts can lead to a decrease in the expression of CD83. The overexpressed SOCS1 transfected on DCs was able to inhibit DC maturation, and the results showed that the expression level of mDCs in mice reinfused with DC-SOCS1 on day 1 was lower than that on day 7. Additionally, it has been reported that CD83 expression is reduced in the airways of COPD patients and is associated with the expression level of TGF-β, which may inhibit the maturation of DCs as well as CD83 expression (18,19). CCR6 is a chemokine receptor expressed on imDCs that regulates the migration of airway imDCs and plays an important role in COPD airway immunity (20). In addition, it has been demonstrated that the expression of CCR6 and CCL20 in induced sputum was upregulated in COPD patients compared to healthy controls and was signi cantly correlated with the severity of the disease (21).
Moreover, we observed the changes of DCs, Th17 and Treg in cigarette smoke-induced COPD mouse models in each group, and the results indicated that in peripheral blood, BALF and lung tissues, the content of mDCs and Th17 were higher in the imDCs and DC-SOCS1 groups, and the content of imDCs and Treg were lower compared with the NS group. Previous studies indicated that Th17 can be induced by cigarettes to produce cytokines such as IL-1β and IL-6, which can inhibit Foxp3 expression and Treg differentiation, and to convert Treg to Th17 (22)(23)(24)(25). Daniela et al. found that Th17/Treg cytokine imbalance induced a worsening of the in ammatory process as well as diffuse structural changes in the lungs in a COPD exacerbation model(26). It suggested that Th17 and Treg mediated immune imbalance contribute to the pathogenesis of COPD.
Th17 has pro-in ammatory activity, and many studies have shown that Th17 and its related cytokines increased signi cantly in COPD patients and COPD animal models, and enhanced the airway in ammatory response by secreting pro-in ammatory factors such as IL-6 and IL-17, which further led to tissue damage(28-30). However, Treg could control the in ammatory response of autoimmune diseases by secreting anti-in ammatory factors such as IL-10, IL-35, and TGF-β (31,32). Consistent with the results of this study, Wang et al. reported that Treg levels were reduced in COPD patients and animal models of COPD compared to controls(28). In this study, we found that the expression levels of IL-23, IL-17, IL-12, IL-21 and IFN-γ were signi cantly decreased in 2×10 6 DC-SOCS1 group than in 1×10 6 DC-SOCS1 and imDCs groups. In particular, the expression levels in group of 2×10 6 DC-SOCS1 on day 1 were lower than in group of 2×10 6 DC-SOCS1 on day 7. However, the concentrations of IL-10, TGF-β, IL-4, and IL-5 were signi cantly higher in the group of 2×10 6 DC-SOCS1 on day 1 than in the group of 2×10 6 DC-SOCS1 on day 7. In addition, the expression levels of IL-10, TGF-β, IL-4 and IL-5 were signi cantly increased in the group with high concentration of DC-SOCS1 than in the group with low concentration and the imDCs group. IL-17 ampli es the in ammatory response by synergizing with various cytokines (37). Treg participates in the immune response and maintains homeostasis together with Th17, mainly by secreting anti-in ammatory factors such as IL-10 to suppress in ammatory responses and maintain immune tolerance(38). SOCS1 can inhibit DCs maturation by suppressing JAK-STAT signaling pathway in DCs cells to maintain DCs stably in the imDCs state (39). It was found that SOCS1 could inhibit the conversion of Treg cells into Th17 cells (5). Similarly, in this study, we found that the secretion of pro-in ammatory factors such as IL-17 and IL-23 were decreased and the secretion of antiin ammatory factors such as IL-10 and TGF-β were increased after injection of DC-SOCS1 compared with the control and imDCs groups. Therefore, the results suggested that DCs transfected with overexpressed SOCS1 could inhibit the secretion of Th17-related cytokines such as IL-17 in COPD.
Moreover, in this study, the content of pro-in ammatory factors such as IL-17 was found to be lower in the 2×10 6 DC-SOCS1 group than in the 1×10 6 DC-SOCS1 group at the same time (day 1 and day 7), while anti-in ammatory factors such as IL-10 and TGF-β increased. The concentrations of pro-in ammatory factors in DCs-SOCS1 injected on day 7 after fumigation were higher than those on day 1, and the concentrations of anti-in ammatory factors decreased. It was suggested that the expression level of in ammatory factors was related to the concentration of injected DCs-SOCS1 and the time of intervention. As IL-10 was involved in the production of regulatory DCs, and overexpression of SOCS1 could induce IL-10 expression on the surface of DCs, so DCs could be converted to regulatory DCs when they produced large amounts of IL-10(5). Therefore, the high concentration of DCs-SOCS1 injected on day 1 compared with day 7 resulted in earlier production of regulatory DCs function. As a result, the secretion of anti-in ammatory factors increased and the secretion of proin ammatory cytokines such as IL-17 was inhibited, and the increase in IL-10 more effectively contributed to Treg production and earlier protection of Treg function, thus further enhancing the inhibition of in ammation in COPD.

Conclusions
In summary, early intervention of COPD mice with high concentrations of SOCS1 lentiviral-transfected DCs can reduce the secretion of pro-in ammatory factors in COPD, attenuate the in ammatory response in COPD, and reduce the risk of death.