FLT3L-mediated CD103+ dendritic cells alleviates hepatic ischemia-reperfusion injury in mice via activation of treg cells

Biomedicine & Pharmacotherapy 118 (2019) 109031

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FLT3/FLT3L-mediated CD103+ dendritic cells alleviates hepatic ischemiareperfusion injury in mice via activation of treg cells Chun-ze Zhoua,b,1, Rui-feng Wangc,1, De-lei Chengb, Yi-jiang Zhub, Qi Caod, Wei-fu Lvb,

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Medical College of Shandong University, Jinan, Shandong, 250021, PR China Interventional radiology department, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, 230001, PR China c Nephrology Department, The Second Affiliated Hospital of Anhui Medical University, Hefei, Anhui, 230001, PR China d Centre for Transplant and Renal Research, Westmead Millennium Institute, University of Sydney, NSW, 2145, Australia b

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Keywords: Dendritic cells Liver Ischemia-reperfusion injury FLT3/FLT3L Treg

Background: This study was conducted to investigate the protective effect of Fms-like tyrosine kinase 3 (FLT3)/ FLT3 ligand (FLT3L)-dependent CD103+ dendritic cells (DCs) on hepatic ischemia-reperfusion injury (IRI). Methods: A mouse model of hepatic IRI and cellular model following hypoxia-reperfusion (H/R) treatment were established. Peripheral blood and liver tissues were obtained and analyzed by flow cytometer in terms of percentage of CD103+DCs and regulatory T (Treg) cells. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were determined to assess liver function. Moreover, proinflammatory cytokines levels including tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 were measured using enzyme-linked immunosorbent assay (ELISA). The histological morphology of liver tissues was examined with hematoxylin and eosin (HE) staining. The apoptosis was detected by terminal deoxynucleotidyl transferase (TdT) dUTP Nick End Labeling (TUNEL) assay. Treg-associated cytokines transforming growth factor (TGF)-β and IL-10 expressions were measured using quantitative real time polymerase chain reaction (qRT-PCR). Results: CD103+ DCs were significantly decreased in peripheral blood and liver tissues of mouse model of hepatic IRI. In vivo experiments indicated that CD103+ DCs infusion ameliorated IRI-induced liver damage and Treg inhibition. Further investigations demonstrated that FLT3/FLT3L-dependent CD103+ DCs suppressed hepatocyte apoptosis via activation of Treg cells in vitro. Conclusion: FLT3/FLT3L-induced CD103+ DCs alleviated hepatic IRI through activating Treg cells.

1. Introduction Ischemia/reperfusion injury (IRI) is a common pathophysiologic process occurring in various clinical settings such as cardiopulmonary bypass and organ transplantation [1]. Particularly, hepatic IRI is characterized by progressive liver injury, hepatocyte apoptosis/necrosis, and acute inflammatory response [2]. Nevertheless, the existing therapeutic outcomes are unsatisfactory, since several experimental studies have demonstrated that the pathogenesis of hepatic IRI was attribute to molecular and cellular pathological processes such as oxidative stress, inflammation, hepatocyte apoptosis, and metabolic disorders, and so on [3]. Therefore, further exploration of the pathogenesis of hepatic IRI will play a crucial role in the development of liver surgery and interventional therapy.

Dendritic cells (DCs) are well known immune cells, which could trigger the innate immune response by secreting various cytokines and chemokines, and induce the adaptive immune response by presenting antigens to T cells [4]. Regulatory T (Treg) cells, a subset of T lymphocytes expressing CD25 and the transcription factor forkhead box P3 (Foxp3), play a key role in maintaining immune homeostasis and inhibiting inflammatory responses by producing interleukin (IL)-10 and transforming growth factor (TGF)-β [5]. Several lines of evidence suggest that highly expressed Treg cells could reduce the inflammation and injury caused by IRI. For example, Gilbert et al [6] reported that preconditioning of Treg cell-depleting antibody reversed kidney IRI-induced neutrophils and macrophages infiltration and partially inhibited the functional and histological injury. Additionally, adoptive transfer of Treg cells, prior to IRI, exerted the protective and anti-inflammatory

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Corresponding author at: Interventional radiology department, The First Affiliated Hospital of USTC, Division of Life Sciences and Medicine, University of Science and Technology of China, No. 17 Lujiang Road, Luyang District, Hefei, Anhui, 230001, PR China. E-mail address: [email protected] (W.-f. Lv). 1 Chun-ze Zhou and Rui-feng Wang contributed equally to this work. https://doi.org/10.1016/j.biopha.2019.109031 Received 28 December 2018; Received in revised form 22 May 2019; Accepted 22 May 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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effects of IRI on the kidney. It is widely accepted that CD103+ DCs, a critical DC subset in parenchymal organs, play a protective immunomodulatory role in various diseases through inducing properties of naïve T cells to Treg cells [7–9]. Our previous study showed that the number of kidney CD103+ DCs was significantly higher in adriamycin nephropathy (AN) mice, whereas depletion of CD103+ DCs attenuated kidney injury in AN mice [10]. However, the underlying mechanism of CD103+ DCs in liver diseases remains unclear. Fetal liver tyrosine kinase 3 (FLT3) is recognized as a member of the type III receptor tyrosine kinase (RTK) family, which is involved in the maturation and differentiation of DCs. CD103+ DCs are mediated by FLT3 through interaction with its cognate ligand (FLT3L) [11]. Collins et al [12] found that FLT3L supplement resulted in a preferential expansion of CD103+ DCs and Treg cells that alleviated the severity of chronic ileitis. Here, we speculated that CD103+ DCs mediated by FLT3/FLT3L pathway induced Treg activation and proliferation, which in turn reduced liver damage and delayed the progression of hepatic IRI. Our study will provide a novel target for molecular targeted therapy of liver IRI.

Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 100 U/ml penicillin and streptomycin (SigmaAldrich, St. Louis, MO, USA) at 37 °C in a humidified atmosphere with 5% CO2. After 24 h culturing, cells were washed with PBS and incubated in serum-free medium in a humidified atmosphere of 1% O2, 5% CO2 and 94% N2 at 37 °C for 12 h. For reoxygenation, cells were later incubated in 5% CO2 for 4 h. 2.4. Isolation and treatment of splenic CD4+ T cells and DCs Spleen tissues of mice were digested by collagenase D (Roche Diagnostics, Meylan, France) for 30 min at 37 °C, and then disrupted using ethylene diamine tetraacetic acid (EDTA; 5 mM; Sigma-Aldrich). The cell suspension was filtered. CD4+ T cells were isolated by the CD4+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA) according to manufacturer’s instruction. For isolation of CD11c+ DCs, total splenocytes after RBC lysis with ACK lysis buffer (Sigma-Aldrich) were incubated with CD11c+ microbeads (Miltenyi Biotec) for 15 min. The cells were then washed, resuspended in cell separation buffer (Dulbecco’s Phosphate-Buffered Saline containing 2% FBS and 2 mM EDTA) and passed through magnetic columns (Miltenyi Biotec) for positive selection. Splenic CD11c+ cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies) containing 10%FBS, 2 mM L-glutamine, 20 mg/ml gentamicin, 100 ng/ml granulocyte-macrophage colony stimulating factor (GMCSF; PEPRO TECH, London, UK), 10 ng/ml IL-4 (Oxford Biomarketing Ltd., Kidlington, Oxford, UK) for 24 h, and then treatemtn with PBS, recombinant FLT3L with or without transfection with siRNA or si-FLT3. The transfection efficiency was verified using quantitative real time PCR (qRT-PCR) and western blotting. CD11c+ DCs receiving the different treatments were used as stimulating cells and CD4+ T cells were used as reactive cells for a mixed lymphocyte reaction (MLR) system. The supernatants were collected for incubation with H/R treated hepatocytes.

2. Materials and methods 2.1. IRI model and sample collection The experimental animals used in this study were male 8-week-old C57BL/6 mice (weighing 20˜25 g), obtained from the Laboratory Animal Center of The First Affiliated Hospital of University of Science and Technology of China. To establish the hepatic IRI model, mice were anesthetized with an intraperitoneal injection of ketamine at a dose of 100 mg/kg as well as 1% pentobarbital sodium at a dose of 10 mg/kg and then kept in the supine position. An abdominal incision was made, and then the perihepatic ligament was dissected to expose the hepatoduodenal ligament. Clamps were used to block the artery/portal vein blood supply to the left and middle liver lobes. After 1.5 h, the clamps were released and the abdomen was subsequently sutured. Mice were placed in a cage for the following 0, 6, 12 and 24 h for reperfusion (n = 8 per group). In addition, sham group (n = 8) performed the identical procedure except vascular occlusion. Mice were sacrificed after reperfusion, and the blood and liver tissue samples were harvested for determining the percentage of various subsets of DCs. All animal procedures were carried out in accordance with the guidelines approved by the Animal Care and Use Committee of The First Affiliated Hospital of USTC.

2.5. Serum biochemical measurements Liver function was assessed by serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) levels measured using a automatic biochemical analyzer (Olympus, Tokyo, Japan) according to the manufacturer's protocol. 2.6. Assessment of pro-inflammatory cytokines levels

2.2. in vivo experiments experiments The levels of various pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-1β and IL-6 were measured using a quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Each blood sample was tested in duplicate.

The IRI groups were then divided into 6 subgroups (n = 8 per group), namely, IRI group, phosphate-buffered saline (PBS) group, tandutinib (MLN518) group, DCs infusion group, MLN518+DCs infusion group, and FLT3L group. Mice in the IRI model group were blocked for 90 min followed by 6 h reperfusion, while PBS group were received PBS (1 mL/day) as the same protocol and schedule. In the MLN518 group, IRI model mice received subcutaneous injections of MLN518 at a dose rate of 20 mg/kg body weight, twice a day for 5 days. In the DCs infusion group, IRI model mice were given intraperitoneal injections of CD103+DCs (5 × 106 cells/mouse) once daily for successive 5 days. In the MLN518+DCs infusion group, combination of MLN518 with CD103+DCs were administered for 5 days before IRI. In the FLT3L group, 5 μg/kg FLT3L were injected intravenously into IRI model mice for 5 days. Peripheral blood and liver tissue samples were obtained for the following experiments.

2.7. Hematoxylin and eosin (HE) staining Liver specimens were fixed with 10% formaldehyde and embedded in paraffin. The specimens were then cut into 4 μm sections and stained with hematoxylin and eosin for histopathologic analysis under light microscopy. 2.8. Isolation of peripheral blood and hepatic CD103+DCs Heparinized peripheral blood samples were mixed with PBS followed by centrifugation for 20 min at 400 g at a room temperature to obtain mononuclear cells. These mononuclear cells were washed with PBS and resuspended in RPMI 1640 medium. DCs were isolated from mononuclear cells using a Blood Dendritic Cell Isolation Kit (Milteyni Biotec) as previously described [13], and seeded in RPMI 1640 medium supplemented with 10%FBS, 2 mM L-glutamine, 20 mg/ml gentamicin,

2.3. Induction of hemorrhage/reperfusion (H/R) in hepatocytes The normal hepatic cell line L-O2 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in 2

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Fig. 1. Changes of DCs subsets in peripheral blood and liver tissues during hepatic IRI. Quantitative analysis of the number of pDCs in peripheral blood (A) and liver tissues (B) of mice in the groups of control, sham, IRI (0 h), IRI (6 h), IRI (12 h) and IRI (24 h). Quantitative analysis of the number of CD103+ CD11b− cells and CD103- CD11b+ cells in peripheral blood (C) and liver tissues (D) of mice. * P < 0.05 vs. sham group; #P < 0.05 vs. IRI (6 h); &P < 0.05 vs. IRI (12 h).

2.10. Quantitative real-time PCR

100 ng/ml GMCSF and 10 ng/ml IL-4 for 24 h. Liver tissues were minced in pre-cooled culture media on ice for preparation of liver cell suspension. The cell pellet was resuspended in 40% Percoll (Amersham Pharmacia, Piscataway, NJ, USA), centrifuged for 20 min at 2,600 g and washed twice with RIPM 1640. After incubation with ACK lysis buffer for 5 min on ice, DCs were cultured in RIPM 1640 supplemented with GMCSF and IL-4 at 37 °C with 5%CO2.

Total RNA was extracted using Trizol reagent (Invitrogen), and reversely transcribed into cDNA using a MiRcute miRNA First-strand cDNA synthesis kit (Tiangen Biotech, Beijing, China) according to the manufacturer’s protocol. The relative quantification of FLT3, TGF-β and IL-10 expression levels were determined using the MiRcute qPCR detection kit (Tiangen Biotech) on an ABI 7500 Real-Time PCR system (Applied Biosystems, Carlsbad, CA, USA) and calculated by the 2−ΔΔCt method. GAPDH served as an internal control.

2.9. Flow cytometry For fluorescence activated cell sorter (FACS) analysis or sorting of DCs, single-cell suspensions derived from peripheral blood and liver tissues were stained with antibodies against class II major histocompatibility (MHC-II), CD11c, F4/80, CD11b, CD103, PDCA and B220 (all form eBioscience, San Diego, CA, USA) and sorted using FACSCalibur flow cytometer (BD Biosciences, San Diego, CA, USA). For flow cytometer analysis of the frequency of Treg cells, the collected CD4+ T cells were washed with PBS and incubated with antiFoxp3 (BD Biosciences) for 30 min at 4 °C. After rinsed twice with PBS, cells were then incubated with FITC-labeled goat anti-mouse IgG (BD Biosciences) for another 30 min at 4 °C in the dark, and analyzed by flow cytometry using FACSCalibur flow cytometer (BD Biosciences).

2.11. Western blot Total protein was obtained using Radio-Immunoprecipitation Assay buffer (Beyotime, Shanghai, China) followed by separated with 6% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes with 350 mA for 4 h. Afterwards, PVDF membranes were blocked with tris buffered saline tween (TBST) containing 5% skim milk at room temperature for 2.5 h and incubated with primary antibodies against FLT3 (#3462; 1:1000; all from Cell Signaling Technology) at 4 °C overnight. Blots were incubated with horseradish peroxidase-conjugated antirabbit secondary antibodies (ab6728; 1:200; Abcam) at room temperature for 1˜2 h and visualized using enhanced chemiluminescence 3

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infusion.

(Thermo Scientific, Shanghai, China) by a Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories, Hercules, CA, USA).

3.3. CD103+ DCs infusion induced Treg activation in mouse hepatic IRI 2.12. Cell apoptosis assay DCs expressing CD103 have been reported to favor the conversion of Foxp3+ Treg cells in the absence of exogenous TGF-β [7]. We thus explored whether CD103+ DCs induced Treg activation in a mouse model of hepatic IRI. As present in Fig. 3, in contrast to CD103+ DCs supplement, MLN518 administration deteriorated IRI-induced the decrease of the percentages of CD103+ DCs and Treg cells and the secretion of Treg-associated cytokines TGF-β and IL-10 in the peripheral blood (Fig. 3A–C) and liver tissues (Fig. 3D–F). On the contrary, FLT3L injection led to an increase in the numbers of CD103+ DCs and Treg cells and the production of TGF-β and IL-10. These findings indicated that CD103+ DCs infusion effectively induced Treg activation in peripheral blood and liver.

Sections were deparaffinized with xylene, eluted by gradient ethanol and washed with PBS, while cells were trypsinized, washed with PBS, and fixed with 4% paraformaldehyde in PBS for 1 h, followed by permeabilization with 0.1% Triton X-100. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction was performed according to the manufacturer’s instructions. The nuclei were stained with DAPI. The stained tissues and cells were examined by an optical microscope (Olympus Corporation, Tokyo, Japan). 2.13. Statistical analysis Data were presented as the mean ± standard deviation (SD) if normally distributed, or as median if otherwise. Student’s t-test and one-way analysis of variance (ANOVA) was used to analyze differences. All P values were two-sided, and P < 0.05 was considered statistically significant.

3.4. FLT3/FLT3L-dependent CD103+ DCs protected hepatocytes against H/R injury via activation of Treg cells Indeed, it has recently been shown that FLT3/FLT3L participates in the pathological process of regulating DCs and Treg cells [14]. We first determined whether CD103+ DCs were FLT3/FLT3L-dependent. The results showed that an increased proportion of CD103+ DCs in splenic DCs were observed following FLT3L treatment, while FLT3 knockdown reversed FLT3L-mediated CD103+ DCs proliferation (Fig. 4A). Transfection efficiency of siRNA was more than 50% after 48 h (Supplementary figure). The MLR demonstrated that FLT3-treated DCs induced increased percentage of Treg cells (Fig. 4B) and production of TGF-β and IL-10 (Fig. 4C) in splenic CD4+ T cells, which were reversed by FLT3 silencing. The effect of FLT3/FLT3L-dependent CD103+ DCs on hepatocyte apoptosis induced by H/R injury was also investigated. The TUNEL-positive apoptotic cells were signally reduced in FLT3L treatment group. Nevertheless, silencing of FLT3 in DCs exerted pro-apoptotic effect via suppressing Treg activation (Fig. 4D). Taken together, these results suggested that FLT3/FLT3L-dependent CD103+ DCs mediated hepatoprotection against IRI via the activation of Treg cells.

3. Results 3.1. Peripheral blood and hepatic CD103+ DCs were significantly reduced in a mouse model of hepatic IRI To characterize the phenotypes of hepatic mononuclear phagocytes (hMPs), we used a combination of markers including CD45, MHC-II, lineage (lin) makers, CD11c, F4/80, CD103, and CD11b. After pregating on CD45+ leukocytes, total hMPs were gated as the lin− MHC-II+ cells, that were further divided into two populations based on their PDCA1 and B220 expression: namely, PDCA+ B220+ cells (plasmacytoid DCs; pDCs) and B220− PDNA+ cells (myeloid DCs; mDCs). IRI induction in mice resulted in a significantly decrease in the numbers of pDCs present in peripheral blood (Fig. 1A) and liver (Fig. 1B) in a time-dependent manner, as compared with those in the sham group. Meanwhile, the lin− MHC-II+ cells were also divided into three populations based on their CD11c and F4/80 expression, including F4/ 80+ CD11c- cells, F4/80+ CD11c+ cells, and F4/80− CD11c+ cells. F4/80− CD11c+ cells were then further divided into two populations based on their CD103 and CD11b expression: namely, CD103+ CD11bcells and CD103− CD11b+ cells. As expected, markedly lower numbers of CD103+ CD11b− DCs, but not CD103− CD11b+ DCs, were found in the peripheral blood (Fig. 1C) and liver tissues (Fig. 1D) of IRI model mice as relative to the sham group.

4. Discussion DCs, as one of antigen presenting cells found in the liver, are divided currently into two broad types: pDCs and conventional dendritic cells (cDCs), that are subdivided further into CD4+ or CD8+ DCs in lymphoid tissues and CD103−/+ DCs in nonlymphoid tissues [15]. Recently, compelling evidence has delineated the role of DCs in hepatic IRI. For instance, Castellaneta et al [16] displayed that interferon (IFN)α derived from liver pDCs played a key role in the pathogenesis of hepatic IRI by enhancing hepatocellular apoptosis, while targeted depletion of mouse pDCs depletion inhibited liver injury and hepatocyte apoptosis and downregulated hepatic IL-6, TNF-α levels. Moreover, Bamboat et al [17] reported that toll-like receptor 9 (TLR9) binding to damage-associated molecular patterns (DAMPs) in cDCs induced IL-10 secretion, thereby restoring liver injury caused by hepatic IRI via reducing the production of proinflammatory cytokines. In addition to antigens presentation, CD103+ DCs display distinct functional activities by conversion of naïve T cells into Treg cells [18]. However, few studies existed with regard to the importance of CD103+ DCs in liverrelated diseases. Heier et al [19] demonstrated that adoptive transfer of CD103+ DCs to Batf3-/- mice of steatohepatitis model could attenuate cellular damage and inflammatory infiltrate. Another research suggested that liver-resident CD103+ DCs served as major antigen presenting cells to induce local CD8+ T cell cytotoxicity in response to hepatotropic viral infection [20]. Based on our finding, the number of peripheral blood and liver-derived pDCs, CD103+ DCs and CD103- DCs were all significantly reduced in experimental hepatic IRI model in mice. Furthermore, adoptive infusion of CD103+ DCs relieved

3.2. CD103+ DCs infusion ameliorated liver injury induced by IRI in mice We then investigated whether CD103+ DCs was required for effective liver protection against IRI. Fig. 2A and B showed that the administration of FLT3 inhibitor MLN518 effectively exacerbated IRI-enhanced ALT, AST, LDH, and the pro-inflammatory cytokines TNF-α, IL1β and IL-6, while CD103+ DCs infusion led to an opposite effect. Likewise, FLT3L injection overturned these effects of MLN518. Consistent with biochemical markers and pro-inflammatory cytokines, HE staining showed that the MLN518 treatment group had similar histologic damage compared with the IRI group, whereas CD103+ DCs infusion and FLT3L administration both alleviated histologic damage (Fig. 2C). To further determine the status of hepatocyte apoptosis, ischemic livers were analyzed by TUNEL staining, which revealed that MLN518 markedly increased the frequency of TUNEL-positive cells compared to the IRI group. Conversely, the number of apoptotic hepatocytes was observably decreased by CD103+ DCs infusion and FLT3L administration (Fig. 2D), supporting the notion that IRI-induced hepatocellular apoptosis was significantly inhibited by CD103+ DCs 4

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Fig. 2. Effect of DCs infusion on IRI-induced hepatocyte apoptosis. Serum ALT, AST and LDH levels (A) and pro-inflammatory cytokines TNF-α, IL-1β and IL-6 (B), HE staining (C) and TUNEL staining (D) of liver tissues obtained from mice in the groups of IRI, PBS, MLN518, DCs infusion, MLN518+DCs infusion, and FLT3L. * P < 0.05 vs. sham group; #P < 0.05 vs. PBS; &P < 0.05 vs. DCs infusion.

Treg are major effector cells in the pathogenesis of hepatic IRI, since Tregs are essential for the maintenance of immune homeostasis in liver inflammatory injury [21]. The percentage of CD4+ Foxp3+ Treg cells was shown to be significantly decreased at early stage of liver IRI. Besides, adoptively transferred Tregs exerted protective effect in hepatic

hepatocellular injury evidenced by serum ALT, AST and LDH reductions as compared to the IRI group. Results of our work also showed significant decreases of all measured serum inflammatory markers, including serum TNF-α, IL-1β and IL-6, coupled with apparent histopathological lesions and hepatic apoptosis after CD103+ DCs infusion. 5

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Fig. 3. Effect of CD103+ DCs on Treg activation in a mouse model of liver IRI. The percentages of CD103+ DCs and Treg cells and the mRNA levels of Treg-associated cytokines TGF-β and IL-10 in the peripheral blood (A–C) and liver tissues (D–F) obtained from mice in the groups of IRI, PBS, MLN518, DCs infusion, MLN518+DCs infusion, and FLT3L. * P < 0.05 vs. sham group; #P < 0.05 vs. PBS; &P < 0.05 vs. DCs infusion.

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Fig. 4. Effect of FLT3/FLT3L-dependent CD103+ DCs on Treg activation and H/R-induced hepatocyte apoptosis. (A) The percentages of CD103+ DCs in splenic DCs following treatment with PBS, FLT3L, FLT3L + siRNA and FLT3L + si-FLT3; The percentages of Treg cells (B) and the mRNA levels of TGF-β and IL-10 (C) in splenic CD4+ T cells following treatment with aforementioned DCs in a MLR system; (D) The TUNEL staining assay in H/ R-treated mouse hepatocytes cultured in MLR system supernatant. * P < 0.05 vs. PBS; #P < 0.05 vs. FLT3L + siRNA.

IRI model [22]. In the present study, we observed that CD103+ DCs infusion increased peripheral blood and liver-derived Treg cells activation and the release of TGF-β and IL-10, suggesting that the immunoregulation between DCs and Treg cells played an important role in the pathogenesis of hepatic IRI. FLT3, that was initially cloned from fetal liver cells with hematopoietic stem cell activity is identified as a key regulatory cytokine for DCs development [23]. DCs can lead to expansion of Tregs by a

mechanism of FLT3/FLT3L signal pathway. Deletion of FLT3 in mice resulted in severe DC deficiency [24]. There is increasing evidence that FLT3/FLT3L participated in the immunoregulatory imbalance between DCs and Tregs. For example, Huang and colleagues [25] indicated that FLT3L-knockout mice showed a significant decrease of the number of pDCs and Treg cells. Additionally, FLT3L treatment significantly expanded Treg, and restored their facilitating function. Our present work demonstrated that FLT3L administration in a mouse model of hepatic 7

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IRI exerted protective effect with evidence of serum ALT, AST, LDH, TNF-α, IL-1β and IL-6 reductions, decreased histopathological lesions and TUNEL-positive apoptotic cells and increased amount of Treg cells and production of TGF-β and IL-10. The percentage of spleen-derived CD103+ DCs were significantly increased along with reduced apoptotic hepatocytes induced by FLT3L treatment, leading to increased Treg cells and TGF-β and IL-10 expression levels, while FLT3 silencing in DCs led to an opposite effect. In summary, we provided the first demonstration of the inhibitory effect of FLT3/FLT3L-mediated DCs in hepatic IRI via activation of Treg cells.

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Conflicts of interest [13]

The authors declare no conflict of interest. [14]

Acknowledgements [15]

This study was funded by Natural Science Foundation of Anhui Province of China(1808085MH254) and Research grant of Tianqing for Liver Diseases(TQGB20180247).

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Appendix A. Supplementary data

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Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109031.

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