Obesity is closely related to exacerbated graft rejection and worse graft survival. High leptin levels, caused by excessive adipose cells in obese individuals, may exert a pivotal role in the pathogenesis of allograft rejection. However, the role and underlying mechanism of leptin in allograft rejection remains unclear. This study explored the role and potential mechanism of leptin in allograft rejection in rats. We performed allogeneic corneal transplantation in rats. The recipients were treated with subconjunctival injections of recombinant rat leptin after transplantation. Clinical evaluation, immunohistological assessment, real-time PCR and flow cytometry were administrated. RAW264.7 macrophages were handled with leptin (3 µg/mL) for 4 hours and then were treated with lipopolysaccharide (10ng/mL) for 24 hours. The culture supernatants were acquired for ELISA. NF-κBp65 activation in RAW264.7 macrophages was detected by western blot. Our results showed that the leptin-treated rats had a significantly reduced corneal graft mean survival time. The number of infiltrating F4/80+CD68+ macrophages increased in the leptin-treated corneal grafts. Monocyte chemoattractant protein-1(MCP-1), tumor necrosis factor-α (TNF-α) and interleukin 6(IL-6) mRNA expression level was higher in the leptin-treated corneal grafts than in the control allografts. Leptin treatment notably increased the frequency and number of T helper 1 (Th1) cells and T helper 17 (Th17) cells in rat ipsilateral cervical lymph nodes. Leptin enhanced NF-κBp65 activation and promoted MCP-1, TNF-α and IL-6 production in RAW264.7 macrophages. Leptin promoted corneal allograft rejection by enhancing the recruitment and activation of macrophages.
Research Article
Leptin Promotes Corneal Allograft Rejection In a Rat Model Via Enhancing Macrophage Activation And Cytokine Production
https://doi.org/10.21203/rs.3.rs-1147201/v1
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Obesity is characterized by increased adipose tissue. Adipose tissue produces numerous adipokines and induces low-grade systemic inflammation that is capable of causing many comorbidities, such as insulin resistance, cardiovascular disease, cancer, asthma and multiple sclerosis [1–4]. In addition, obesity aggravates cardiac allograft rejection by enhancing the capacity of antigen-presenting cells (APCs) [5]. However, the exact immune mechanism by which obesity accelerates allograft rejection is unclear.
Recently, experimental studies have proposed that proinflammatory adipokines in obesity may affect alloreactivity, accelerating acute rejection [5]. Among these adipokines, leptin as a 16-kDa non-glycosylated hormone mainly produced by adipose cells, plays a crucial role in linking metabolism and the immune response[6]. High leptin levels secreted by the excessive adipocytes induce a low-grade inflammatory condition that causes obese individuals to be more susceptible to autoimmune diseases, such as multiple sclerosis, inflammatory bowel disease, autoimmune diabetes, psoriasis, autoimmune thyroiditis, and rheumatoid arthritis[7]. Indeed, leptin deficiency has been reported to reduce alloreactivity and prolong allograft survival after skin transplantation in mice [8]. It was indicated that leptin had the capacity to promote the corneal neovascularization in rats[9]. However, the role of leptin on corneal allograft rejection is still elusive.
It has been reported that macrophages are closely involved in allograft rejection [5]. Depletion of macrophages reduces tissue injuries and acute rejection in rats, demonstrating that macrophages are important effector cells during acute renal allograft rejection [10]. Similarly, macrophages are also involved in the pathological process of corneal allograft rejection [11]. Macrophages could present antigens to CD4+ T cells and to secrete proinflammatory cytokines that can up-regulate MHC class-II molecules and co-stimulatory molecules, accelerating corneal allograft rejection [12].
Since leptin has been shown to modulate macrophage functions [13], we speculate that leptin participates in allograft rejection pathogenesis by promoting macrophage function. Obese rats showed systemic metabolic abnormalities. Here, in order to determine the effects of leptin on allograft survival, we use subconjunctival leptin injections in the rat model of corneal transplantation.
Animals
The studies were approved by the Institutional Animal Care and Use Committee of the Zhongshan Ophthalmic Center at Sun Yat-sen University. Female Sprague-Dawley rats (200 to 250 g, 6-8 weeks old) were served as recipients for the allograft, and Wistar rats (200 to 250 g) were used as donors. The rats were feeded under the condition (a 12-hour light/dark cycle at 23 ◦C ±2 ◦C and 55% ±10% humidity).
Penetrating Corneal Transplant Surgery
The rats were anesthetized by intraperitoneal injection of pentobarbital. Central, full-thickness corneas were excised from donors with a 3.5-mm-diameter trephine. These buttons were transplanted onto a 3-mm-diameter corneal bed in each recipient right eye via eight discontinuous sutures (10-0 nylon, Alcon, Fort Worth, Texas, USA). To test for the correct transplantation technique, autograft corneal transplantation were performed. Recombinant rat leptin (10 µg in 20 µL of phosphate buffer saline [PBS], R&D Systems, Minneapolis, Minnesota, USA) was injected subconjunctivally at the time of operation and on days 3, 5 and 7 postoperatively. In the control group, the grafted recipients were executed by subconjunctival injections with 20 µl PBS. The corneal grafts were observed by a slit lamp biomicroscope three times per week.
Clinical Evaluation
The grafts were assessed clinically by using the scoring system (RI) [14]. The following scoring system was used for opacity: 0, transparency; 1, mild opacity, iris are apparently obsrvable; 2, a few details of iris are sightless; 3, details of iris are sightless, but pupil is distinguishable; and 4, completely opacity. Edema was evaluated by the scores: 0, no edema; 1, slight edema; 2, middling edema; and 3, remarkable edema. Neovascularization was evaluated by the following scores: 0, devoid of vessels; 1, vessels developing in the graft bed; 2, vessels growing at the graft edge; 3, vessels developing deeper; and 4, vessels appearing in the central region of graft. The graft was marked to be rejected when RI score was greater than 5.
Histology
The rats of each group were killed on days 9 after surgery. Briefly, the eyes were fixed in 10% (V/V) formalin overnight, transferred to increasing concentrations of ethanol, and paraffin-embedded. Corneal tissue was cut into 3-µm-thick sections, was dewaxed three times in xylene for 30 minutes, and then was rehydrated. Slides were incubated in hematoxylin for 1 minute, followed by washing in water for 1 minute. Then, the slides were placed into 1% hydrochloric acid for 30 seconds, washed in water for 20 minutes, stained in eosin for 1 minute, washed again in water for 2 minutes, dehydrated through increasing concentrations of ethanol, mounted and coverslipped. The number of inflammatory cells in the grafts was enumerated (n=3 random samples, 400× magnification), and the numbers from all fields were averaged.
Fluorescence Immunohistochemistry
Corneal grafts of each group were embedded after fixation and then cut into 8-µm-thick slices on days 9 postoperatively. For fluorescence immunohistochemistry, the slices were washed with PBS for 15 minutes. After blocking for 60 minutes, the slices were incubated with the following primary antibodies: mouse anti-CD68 antibody (1:200, Abcam, Cambridge, UK) and chicken anti-F4/80 antibody (1:200, Abcam). The slices were washed for 30 minutes and then stained with the following antibodies: FITC-conjugated donkey polyclonal anti-chicken antibody (1:500, Abcam) and AlexaTM 568-conjugated donkey anti-mouse IgG (1:500, Invitrogen, Carlsbad, California, USA). The stained slides were detected by a laser confocal microscope (LSM700; Carl Zeiss MicroImaging GmbH, Jena, Germany). The number of F4/80+ and CD68+ cells in the corneal grafts was enumerated (n=4 random samples, 200× magnification), all fields were averaged, and the number of macrophages was reported.
Real-time Quantitative Polymerase Chain Reaction (RT-PCR)
The corneal allografts of each group were dissected from the rats and lysed in TRIzol reagent (Invitrogen, USA). The corneal pieces were sonicated for 5 minutes. Total RNA was extracted in accordance with the standard instructions. Real-time PCR (RT-PCR) was performed using the PrimeScriptTM RT reagent kit (Takara, Osaka, Japan) following the instructions.The following primers were used for qPCR in table 1：GAPDH, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) [15].
Flow Cytometry
The cell suspensions were acquired from recipient ipsilateral cervical lymph nodes on days 10 postoperatively. To detect the intracellular cytokines in T lymphocytes, the cells were treated with phorbol myristate acetate (50 ng/ml; Sigma, St. Louis, MO, USA) and ionomycin (500 ng/mL; Sigma) and then blocked with brefeldin A (1 µg/ml; Sigma) under the condition of 37 °C and 5% CO2 for 5 hours. The collected cells were incubated with anti-rat CD4 APC (BD Biosciences, USA), anti-rat IFN-γ FITC (BD Biosciences) and anti-rat IL-17A PE-Cy7 (eBioscience) antibodies for T helper 1 (Th1) and T helper 17 (Th17) cell analysis. The cells were determined using flow cytometer (BD Biosciences). The data were further evaluated by FlowJo 10 software.
Cell Treatment
RAW264.7 macrophages were obtained from ATCC and were cultured in culture media with 10% heat-inactivated fetal bovine serum (FBS, Gibco, Carlsbad, California, USA). The RAW264.7 macrophages were cultured in 24-well plates (2×105 cells/well) with 0.5 ml culture media containing 0.5% FBS overnight. The medium was exchanged with new medium with or without leptin (3 µg/mL) for 4 hours; then, the cells were handled with 10 ng/mL lipopolysaccharide (LPS, Sigma) for an additional 24 hours. Culture supernatants were acquired for enzyme-linked immunosorbent assay (ELISA).
ELISA for Cytokine Quantification
Monocyte chemoattractant protein-1(MCP-1), tumor necrosis factor-α (TNF-α) and interleukin 6(IL-6) levels in the supernatants were detected by mouse “sandwich” ELISA kits (eBioscience, San Diego, California, USA) following the specifications.
Western Blot Analysis
After stimulation by leptin for 4 hours and/or LPS for 30 minutes, RAW264.7 macrophages were dissolved in RIPA lysis buffer and then centrifuged (14,000×g) for 15 minutes. The supernatants were obtained. A total of 30µg protein was separated by 10% SDS-PAGE, transferred to PVDF membrane and treated with the primary antibodies against GAPDH, NF-κBp65 and phospho-NF-κB p65 (1:1000，Cell Signaling Technology, Boston, Massachusetts, USA) at 4 ◦C overnight. The PVDF membrane then was treated with HRP anti-rabbit IgG (1:5000, Cell Signaling Technology) for 1 hour at indoor temperature.
Statistical Analysis
The results are displayed as means ± SD. Corneal rejection was measured by the Kaplan-Meier estimator and log-rank test. Statistical significances were determined by an independent unpaired two-tailed Student tests or one-way ANOVA. P <0.05 was considered as significant.
Leptin Aggravated Corneal Allograft Rejection in Rats
The autografts and untreatment allografts were observed for 60 days. All autografts survived for at least 60 days (Figure. 1A). The mean survival time of untreatment allografts were 12.38±1.5 days (Figure. 1A). Corneal graft rejection in the control rats occurred within 11 to 15 days after transplantation, averaging 12.07±1.32 days (Figure. 1B). In the leptin-treated group, the grafts were rejected within 9 to 11 days, averaging 9.92±1.04 days (Figure. 1B). In comparison with that of the control group, corneal graft rejection was exacerbated by subconjunctival leptin injections in the treatment group (Figure. 1B). The difference in allograft survival time between the control group and leptin-treated group was significant (P<0.001, Figure. 1B). The rejected allografts in the leptin-treated group displayed significant opacification, edema, and neovascularization, which appeared in the graft periphery on days 9 after transplantation; on the contrary, the nonrejected allografts in the control group presented with slight opacification (Figure. 1C). The averaged RI, opacity, edema, and neovascularization scores on days 9 were 6.08±1.12, 2.92±0.86, 1.46±0.52, and 1.54±0.52, respectively, for allografts in the leptin-treated group and 4.38±0.77, 2.08±0.64, 1.08±0.28, and 1.31±0.48 in the control group (Figure. 1D-G, P<0.01, P<0.01, P<0.01, and P>0.05, respectively).
Leptin Enhanced Inflammation in Corneal Grafts
Hematoxylin-eosin staining in the corneal allografts on days 9 showed that the number of infiltrating inflammatory cells and the amount of apparent stromal fiber edema increased in both the control and the leptin-treated groups compared with the group with normal, ungrafted corneas (Figure. 2A). Compared to the control grafts, the leptin-treated grafts displayed an increase in central corneal thickness and inflammatory cell number (Figure. 2B, C, P<0.01, P<0.01).
Leptin Promoted Macrophage Recruitment in the Corneal Allografts
To detected infiltrating macrophages on days 9, we stained corneal allografts with F4/80 and CD68. The F4/80+ CD68+ cells were considered to be macrophages. In contrast to the control allografts, the leptin-treated grafts displayed more macrophages (Figure. 3A, B, P<0.01).
Leptin Up-regulated Immune Factor Expression in vivo
MCP-1 is a critical chemoattractant which attracts macrophages to corneal grafts following transplantation [16]. In return, macrophages produce abundant MCP-1, causing inflammatory cells to infiltrate the corneal allografts [17]. On days 9, MCP-1 mRNA expression was enhanced in the leptin-treated corneal allografts compared to the control allografts (Figure. 4A, P<0.05).
TNF-α and IL-6 are the crucial cytokines that is mainly secreted by macrophages. In our results, compared to the normal corneas, the mRNA expression of TNF-α and IL-6 increased in the corneal allografts on days 9 (Figure. 4B, C, P<0.001 and P<0.01). The mRNA expression of TNF-α and IL-6 was substantially higher in the leptin-treated corneal allografts than in the control allografts (Figure. 4B, C, P<0.05 and P<0.01).
Leptin Promoted Th1 and Th17 Differentiation in Rat Ipsilateral Cervical Lymph Nodes.
Effective T cells (Teffs) in draining lymph nodes, specifically Th1 and Th17 cells, were involved closely in the immunopathogenesis of corneal allograft rejection [18-20]. Therefore, we explored the effects of leptin on Th1 and Th17 cell populations in rat ipsilateral cervical lymph nodes. The results indicated that leptin significantly increased the frequency and number of CD4+ IFN-γ+ Th1 cells and CD4+ IL-17A+ Th17 cells compared with the control group (Figure. 4A-F, P<0.01 and P<0.01).
Leptin Promoted Immune Factor Secretion from Macrophages in vitro
The MCP-1, TNF-α and IL-6 secretion from RAW264.7 macrophages was significantly promoted by leptin compared with the LPS-stimulated group alone (Figure. 5D-F, P<0.01, P<0.05 and P<0.05). We found that leptin amplified the macrophage response to LPS, which then increased MCP-1, TNF-α and IL-6 production.
Leptin Enhanced the Activation of NF-κBp65 in Macrophages
NF-κB signal pathway exerted a pivotal role in the production of proinflammatory cytokines in macrophages[21, 22]. In the study, we found that NF-κBp65 phosphorylation was enhanced after LPS-stimulation in RAW264.7 macrophages (Figure.6 , P<0.01). Leptin augmented the activation of NF-κBp65 stimulated by LPS (Figure.6, P<0.05).
Obesity is gradually becoming a major public health concern, and the prevalence of this disease has been increasing worldwide over the last few decades [23]. Recently, researchers have found that increased leptin levels, due to patient adipose tissue, may be related to allograft failure [24] but the underlying mechanism by which leptin affects allograft rejection is unclear. In this study, using subconjunctival leptin injections, we identified the effects of leptin on corneal allograft rejection in rats. In comparison with the control group, leptin significantly exacerbated corneal allograft rejection in rats. The results displayed that leptin promoted MCP-1, TNF-α and IL-6 mRNA expression, increased the number of macrophages in corneal grafts and enhanced the populations of Th1 and Th17 cells in rat ipsilateral cervical lymph nodes.
It was reported that systemic injection of leptin significantly reduced the caloric intake and body weight, which will affect the immune system in rats [6, 25, 26]. To study the direct effects of leptin on local tissues, leptin injections locally into ventricles, stellate ganglions and knee joints were used successfully in various animal models [27–30]. Therefore, we used subconjunctival injections of recombinant leptin to explore the effects on macrophages after corneal allograft transplantation.
Corneal transplantation is the main treatment for corneal blindness. Corneal rejection is the main reason of transplant failure[31]. Previous studies have indicated that the immune system causes corneal transplant rejection [32]. Among the many factors that cause corneal rejection, macrophages play a critical role in this acute process [33]. Corneal rejection can be effectively prevented by subconjunctival injections of dichloromethylene diphosphonate, which depletes local macrophages without directly disturbing other immune cells [11]. An increasing amount of evidence indicates that macrophages function as antigen-presenting cells to induce adaptive immune cell activation. Furthermore, macrophages also produce chemokines and proinflammatory cytokines, including MCP-1, TNF-α, IL-6, interleukin-1β (IL-1β), and interferon-γ (IFN-γ), to promote corneal allograft rejection [34, 35].
In innate immunity, leptin facilitates the secretion of TNF-α, IL-6 and IL-1β from human mononuclear cells [36]. Inflammatory cytokine production in macrophages are closely related to the activation of NF-κ B [37]. Indeed, we also found that leptin enhanced the activation of NF-κBp65 in macrophages. Leptin deficiency impairs macrophage phagocytosis and decreases proinflammatory cytokine expression, such as TNF-α, IL-6, and interleukin-12 (IL-12), in vivo and in vitro [6]. Our current findings also indicate that leptin can promote proinflammatory cytokine secretion in macrophages, such MCP-1, TNF-α and IL-6. In addition, MCP-1, TNF-α and IL-6 mRNA expression was increased significantly in the leptin-treated corneal allografts compared with the control allografts.
MCP-1 is a primary chemotactic factor in mononuclear cell recruitment, and MCP-1−/− mice cannot recruit macrophages in several different animal models [16, 38]. In a corneal inflammation model, MCP-1 deficient mice had fewer macrophages infiltrating the inflammatory site of the cornea [16]. This results suggests that MCP-1 is necessary for macrophage recruitment but not for the recruitment of dendritic cells or neutrophils [16, 38]. It was demonstrated that the expression of MCP-1 is higher in high-risk corneal grafts than in control grafts, which recruits additional macrophages on days 6 after transplantation in mice [17]. Our findings indicate that leptin also up-regulates MCP-1 expression in vivo. Indeed, on days 9 after transplantation, we detected an increase in macrophage infiltration in the leptin-treated corneal grafts. Therefore, leptin enhances the recruitment of macrophages to corneal grafts by increasing the expression of MCP-1 in the corneal grafts.
TNF-α is mainly derived from the monocyte/macrophage lineage. The TNF-α mRNA level was significantly increased in allografts during corneal rejection, but a low expression level was detected in the control autografts [39]. The TNF-α level in the aqueous humor of recipients with rejected corneal allografts is substantially increased [40]. These experiments indicate that TNF-α is closely associated with corneal rejection in allograft transplantation. Previous studies have revealed that TNF-α induces apoptosis in corneal epithelial cells, keratocytes, and endothelial cells [41, 42] and destroys the barrier integrity of the corneal endothelium [40]. TNF-α-induced apoptosis plays an important role in endothelial injury during corneal allograft rejection [41, 42]. In addition, TNF-α has the capacity to increase the number of langerhans cells situated at the corneal limbus and to induce a dose-dependent effect on corneal langerhans cell migration into the central cornea, which exacerbates the allograft rejection [43]. Intracameral injections of a recombinant TNF-α receptor inhibitor that blocked TNF-α activity significantly prolonged corneal allograft survival after corneal transplantation [44]. Our data indicate that leptin promotes the secretion of TNF-α in macrophages after LPS stimulation in vitro and enhances TNF-α mRNA expression in vivo.
It was widely believed that Th1 and Th17 cells from draining lymph nodes induced corneal graft rejection [18–20]. In the study, our findings indicated that leptin significantly increased the percentage of Th1 and Th17 cells in rat ipsilateral cervical lymph nodes. Therefore, we suggest that subconjunctival injections of leptin promote Th1/Th17 differentiation in draining lymph nodes in rats by enhancing the functions of macrophages.
We show that leptin promotes Th1/Th17 differentiation in draining lymph nodes and then accelerate corneal allograft rejection via enhancing macrophage activation and cytokine production. Leptin may be a potential therapeutic target for delaying corneal graft rejection. Further research is necessary to identify the effects of increased leptin levels on the transplantation of other organs.
Statements and Declarations
Funding
This work was supported by Science and technology cooperation project of Hong Kong, Macao and Taiwan (2015DFH30190); The Science and Technology Project of Nantong Municipality (JCZ19009).
Author contributions
All authors made a contribution to the conception and design of the study. JFY, XQC, YQL and FT performed the experiments and analyzed the data. The manuscript was written and edited by JFY, WRS and DL. All authors read and approved the submitted manuscript.
Data Availability
The data supporting the conclusion are included in the article.
Compliance with ethical standards
Ethics approval
All animal procedures performed in this study were approved by the Institutional Animal Care and Use Committee of the Zhongshan Ophthalmic Center at Sun Yat-sen University (protocol number: 2016113).
Competing interests
The authors declare that they have no competing interests.
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Table 1 Primer sequences
