A Novel Immunosuppressant, Luteolin, Modulates Alloimmunity and Suppresses Murine Allograft Rejection

Immune-based allograft rejection is a main reason for graft failure after transplantation. The progress of allograft rejection is associated with the activation of alloimmune responses largely mediated by effector T cells (1–4). Immunosuppressive regimens, including corticosteroids, calcineurin inhibitors, and inhibitors of mammalian target of rapamycin (mTOR), are widely used for prevention of both acute and chronic transplant rejection. However, transplant survival may be compromised due to adverse reactions caused by a conventional immunosuppressant (5–8). Current immunosuppressive measures may lead to renal dysfunction, cardiovascular toxicity, and increased incidence of cancer or infection (9, 10). An ideal immunosuppressive protocol is to induce long-term transplant acceptance or tolerance with minimal adverse reactions. Natural ingredients, derived from herbal medicine or edible plants, have been reported to be effective in suppression of allograft rejection with little adverse reactions in experimental animal models (11–13), although it remains to be determined if a natural ingredient generates less side effects or toxicity than a conventional immunosuppressant.

Luteolin, a plant flavonoid, is commonly present in many herbs and vegetables, including chrysanthemum, honeysuckle, dandelion, pepper, broccoli, and celery. Luteolin exhibits multiple biological and pharmacological effects, such as anti-inflammatory (14–16), anticancerous (17, 18), and neuroprotective properties (19, 20). Recent studies have reported that luteolin suppresses the expression of proinflammatory cytokines, including TNF-α, IL-6, IFN-γ, and IL-5 (20, 21). Importantly, luteolin has been shown to promote CD4⁺CD25⁻ T cell differentiation into CD4⁺CD25⁺ regulatory T cells (Tregs) in the context of airway inflammation (22). Moreover, a previous study has revealed that renal ischemia/reperfusion injury after kidney transplantation is improved by administration of luteolin (23). However, it is unknown whether luteolin regulates alloimmunity and suppresses allograft rejection. In contrast, mTOR signal pathway is generally acknowledged as a negative regulator of CD4⁺Foxp3⁺ Treg development (24). Given that luteolin has been shown to upregulate Tregs and suppress inflammation, we hypothesized that luteolin could be an mTOR inhibitor that might inhibit allograft rejection by inducing CD4⁺Foxp3⁺ Tregs.

In this study, we used a mouse model of skin allotransplantation to determine the effects of luteolin on allograft rejection and to identify its mechanisms of action. Our results demonstrated that luteolin significantly prolonged skin allograft survival, ameliorated cellular infiltration, and downregulated proinflammatory cytokine gene expression in skin allografts. Further, luteolin increased the frequency of CD4⁺Foxp3⁺ Tregs while reducing the expansion of mature dendritic cells (DCs) and CD44^highCD62L^low effector CD4⁺/CD8⁺ T cells posttransplantation. Luteolin also suppressed T cell proliferation and induced CD4⁺Foxp3⁺ Tregs in vitro. Moreover, luteolin inhibited AKT/mTOR signaling in T cells in vitro. Thus, we demonstrated that luteolin is, to our knowledge, a new immunosuppressant as an mTOR inhibitor in the context of allotransplantation.

Six- to eight-week-old male C57BL/6 and BALB/c mice (body weight: 20 ± 2 g) were obtained from Guangdong Medical Laboratory Animal Center (Guangzhou, China) and were housed under a specific pathogen-free condition. The animal protocol was approved by the Institutional Animal Ethical Committee of Guangdong Provincial Academy of Chinese Medical Sciences. All animal experiments were strictly executed in accordance with the national guidelines.

BALB/c mice were skin donors, whereas skin graft recipients were C57BL/6 mice. Full-thickness trunk skin sized ∼1 × 1 cm² was transplanted to the right dorsal flank area of recipient mice and secured with a sterile bandage. Skin graft was monitored daily after removal of the bandage 8 d after transplantation. Skin allograft rejection was defined as graft necrosis larger than 90%, as we described previously (25, 26).

Mice were randomly divided into four groups, including control group (by mouth, 0.5% CMC-Na; Sigma), rapamycin (Rapa) group (i.p. Rapa, 1 mg/kg; Sigma), and luteolin group (by mouth, low dose of luteolin [Lut-low] 25 mg/kg or high dose of luteolin [Lut-high] 50 mg/kg, purity >98%; Daosifu Bio-Technique), and treated daily for consecutive 3 wk or until graft rejection/sample collection. Luteolin was prepared with 0.5% CMC-Na solution, whereas Rapa was dissolved in saline. Control groups were given equivalent 0.5% CMC-Na solution only. In our previous study, we have revealed that oral administration of 0.5% CMC-Na in recipient mice did not alter allograft rejection and Treg frequency compared with totally untreated mice. To deplete CD4⁺CD25⁺ Tregs, mice were treated with anti-CD25 Ab (PC61; eBioscience) at 0.2 mg on days 0, 3, and 6 posttransplantation.

Skin grafts were removed from euthanized recipient mice, fixed with 4% paraformaldehyde for 24 h, and embedded in paraffin after dehydration. Paraffin sections of 3-μm thickness were then made, deparaffinized, and stained with H&E. For immunohistochemistry, sections were incubated first with primary anti-CD3 Ab (1:100; Abcam) at 4°C overnight after Ag retrieval, then counterstained with secondary Ab HRP–anti-rabbit IgG (Maxim) at 37°C for 30 min, and finally colored with diaminobenzidine at room temperature. For quantitative analysis, slides were imaged under 100× light microscope fields. The inflammatory cell infiltration and integrated OD of CD3-positive staining in the image was measured using image-processing software (ImageJ 1.47).

Skin allografts of recipient mice were harvested, embedded in OTC, and frozen. Skin tissues were cut into sections (3 μm) using freezing microtome. Then, they were blocked with 0.3% Triton X-100 and 10% BSA for 1 h. Subsequently, the sections were incubated with primary rabbit anti-Foxp3 (Cell Signaling Technology) Ab at a concentration of 1:100 at 4°C overnight. Upon being washed with PBS, sections were stained with a secondary Ab anti-rabbit IgG conjugated with Alexa Fluor 488 (Cell Signaling Technology). These cryosections were finally mounted using DAPI-Fluoromount-G clear mounting agents (Southern Biotech). The sections were observed using a fluorescence microscope (original magnification ×200), and the photomicrographs were merged through Adobe Photoshop. The fluorescence intensity in all of the images was measured using ImageJ 1.47 software.

Draining lymph node (LN) cells or splenocytes were isolated from recipient mice and stained for surface markers with anti-CD4–FITC (clone H129.19), CD8-FITC (clone 53-6.7), CD44-V450 (clone IM7), and anti-CD62L–allophycocyanin (clone MEL-14) Abs (BD Biosciences and eBioscience). For intracellular Foxp3 analysis, cells were fixed and permeated using Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent Kits (eBioscience). Then, cells were stained with anti-Foxp3–allophycocyanin mAb (clone FJK-16s; eBioscience). The frequencies of CD4⁺Foxp3⁺ Tregs and CD44^highCD62L^low effector T cells were finally analyzed using a flow cytometer (FACSCalibur; BD Biosciences). To isolate CD3⁺ and CD4⁺CD25^– T cells, splenocytes were stained with anti-CD3–allophycocyanin (clone 145-2C11), CD4-FITC (clone H129.19), and anti-CD25–PE (clone 3C7) Abs (BD Biosciences), and then sorted via FACSAria III (BD Biosciences). The purity of the sorted cells was >96%.

FACS-sorted CD4⁺CD25^– T cells from naive C57BL/6 mice were cultured in 96-well plates (200 μl/well) at 5 × 10⁵ cells per well in complete RPMI 1640 medium (Gibico) supplemented with 10% FBS (Gibico) and stimulated with anti-CD3/anti-CD28 Abs (2.5 μg/ml, GER; Sigma) plus IL-2 (10 ng/ml; PeproTech) in the absence or presence of luteolin (Lut-low: 2.5 μM or Lut-high: 5 μM) for 4 d. Finally, FACSCalibur was used to determine the frequency of CD4⁺Foxp3⁺ Tregs.

FACS-sorted CD3⁺ T cells derived from C57BL/6 mice were incubated with CFSE (3 μM; Invitrogen) for 15 min at room temperature without light. Then, cells (4 × 10⁵ cells per well) were cultured in complete RPMI 1640 medium (200 μl; Gibico) in 96-well plates in the presence of anti-CD3/anti-CD28 Abs (2.5 μg/ml; Sigma) and rIL-2 (10 ng/ml; PeproTech). These cells were also treated with luteolin (Lut-low: 2.5 μM or Lut-high: 5 μM) or Rapa (0.1 μM). Four days later, cell proliferation was measured using FACSCalibur (BD Biosciences), whereas the expression levels of IFN-γ, IL-10, and IL-17a in the supernatant were detected via ELISA according to the manufacturer’s protocol (Boster).

Cytotoxicity to T cells was detected using CCK-8 assays. Briefly, FACS-sorted CD3⁺ T cells derived from C57BL/6 mice were seeded at a density of 2 × 10⁶ cells/ml (4 × 10⁵ cells per well in 200 μl) in 96-well plates and cultured in complete RPMI-1640 medium (Gibico) containing 10% FBS (Gibico) in the presence of anti-CD3/anti-CD28 Abs (2.5 μg/ml) and rIL-2 (10 ng/ml; PeproTech). Luteolin was added to each well at a series of concentrations (1.25, 2.5, 5, 10, and 20 μM, respectively) in quadruplicate. After 48 or 96 h, 20 μl of CCK-8 was added to each well of the culture that was further incubated at 37°C for 4 h. The absorbance at the wavelength of 450 nm was measured using a microplate spectrophotometer (Thermo Fisher Scientific). Control value without luteolin was set as 1.0.

Total mRNA from graft tissue was extracted using TRIzol reagents (Invitrogen). RNA concentration was determined according to absorbance at 260 nm, whereas purity was evaluated based on the ratios of A260/A280. mRNA was then reversely transcribed to cDNA with PrimeScript RT reagent kits (Takara Bio) according to the instructions of the manufacturer. The cDNA was analyzed for the expression of cytokines using a Quantifast SYBR Green PCR kit (TAKARA Bio) by an ABI 7500 Fast Real-Time PCR System (Thermo Fisher Scientific). β-Actin was used to determine relative expression of target genes, and analysis was performed through a comparative 2^△△CT method. The primer sequences are listed in Table I.

T cells were lysed by RIPA lysis buffer, and concentration of protein was detected by a BCA protein assay kit (Thermo Fisher Scientific). Protein of each sample was run on 10% SDS-PAGE gels and transferred into a PVDF membrane. After blocking in TBST with 5% (w/v) BSA at room temperature for 1 h, the membrane was incubated with rabbit primary Abs, including anti–P-mTOR and anti-mTOR, anti–P-p70S6K, anti-p70S6K, or anti-GAPDH Abs (1:1000; Cell Signaling Technology) at 4°C overnight, washed, and then incubated with a secondary Ab, HRP-conjugated goat anti-rabbit IgG (1:2000) for another hour. Finally, the signals were detected using an ECL method (Beyotime, China) and analyzed via ImageJ software (NIH Image).

Comparisons of means were performed using Student t test and one-way ANOVA. Data were presented as mean ± SD and analyzed via GraphPad Prism 6 (GraphPad Software). The analysis of graft survival was conducted using the Kaplan–Meier method (log-rank test). A comparison of difference was considered statistically significant when a p value was <0.05.

Because luteolin can regulate immunity, we asked whether luteolin would suppress allograft rejection. C57BL/6 mice were transplanted with skin from donor BALB/c mice and treated with luteolin or Rapa. As shown in Fig. 1A, graft photos were representatives of skin allografts in C57BL/6 mice that were untreated or treated with luteolin or Rapa 14 d after transplantation. There was no sign of rejection in mice treated with Rapa or Lut-high, whereas rejection obviously occurred in a control recipient. As also shown in Fig. 1B, we found that treatments with both Lut-low and Lut-high prolonged skin allograft survival compared with the control group (median survival time = 22 [Lut-low] versus 16 d [control group], p < 0.05; and 35 [Lut-high] versus 16 d [control group], p < 0.01). Interestingly, when compared with Rapa (Rapa), Lut-high treatment exhibited a nearly equivalent effect on prolongation of skin allograft survival (median survival time = 33 versus 35 d, p > 0.05). Histologic appearance of graft rejection is generally accompanied by extensive infiltration of leukocytes. Because luteolin delayed allograft rejection, we examined cellular infiltration in an allograft via H&E and immunohistochemical staining 10 d posttransplantation. As shown in Fig. 1C, a significant decrease in cellular infiltration was observed in both luteolin- and Rapa -treated groups versus control group (p < 0.01). In contrast, CD3⁺ T cells play an important role in allograft rejection (4). Our immunohistochemical results revealed that luteolin, at either low or high doses, also suppressed CD3⁺ T cell infiltration in skin allografts of the recipient mice compared with the control group (Fig. 1D).

As luteolin reduced CD3⁺ T cell infiltration in a skin allograft, we assumed that it would regulate effector T cells in vivo. Ten days after skin transplantation, C57BL/6 mice were treated with luteolin or Rapa, and draining LN and spleen cells were isolated. The frequencies of CD44^highCD62L^low effector CD4⁺ or CD8⁺ T cells were analyzed by FASC analyses. Compared with the control group, luteolin at either low or high doses significantly reduced the frequencies of CD4⁺CD44^highCD62L^low or CD8⁺CD44^highCD62L^low effector T cells in both LNs and spleens (Fig. 2). These results suggest that luteolin suppresses the generation of CD44^highCD62L^low effector T cells in the context of allotransplantation.

Our data demonstrated that luteolin alleviated cellular infiltration in the skin allografts. Thus, we further asked whether luteolin would regulate proinflammatory cytokines. Total mRNA in skin allografts was extracted, and mRNA expressions of IFN-γ, IL-6, IL-10, TNF-α, IL-17A, and Foxp3 were determined by RT-PCR 10 d after skin transplantation and luteolin treatment. The primer sequences are listed in Table I. As shown in Fig. 3, luteolin, at both doses, downregulated gene expressions of proinflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-17A, whereas Foxp3 expression was upregulated by luteolin. Further, Lut-high increased IL-10 gene expression. Our findings suggest that luteolin indeed suppresses gene expressions of major proinflammatory cytokines while increasing mRNA level of immunosuppressive cytokine IL-10.

To investigate whether luteolin would also interfere with DC maturation amid alloimmune responses and transplant rejection, splenocytes and draining LN cells were isolated and analyzed for the markers of DC maturation 10 d after skin allotransplantation. As shown in Fig. 4A, either Lut-low or Lut-high significantly lowered the percentage of CD86⁺ mature DCs within CD11c⁺ population in both spleens and LNs. Moreover, Lut-high reduced the frequency of CD11c⁺ DCs. However, when only CD11c⁺CD86⁺ mature DCs were gated, the expression level (median fluorescence intensity [MFI]) of CD86 was no longer affected by luteolin, suggesting that luteolin mainly reduces the frequency of CD86⁺ mature DCs but not their relative expression level. Similarly, luteolin decreased the frequency of CD80⁺ mature DCs within CD11c⁺ population in both draining LNs and spleens, whereas luteolin also did not alter the expression level (MFI) of CD80 when only CD11c⁺CD80⁺ mature DCs were gated (Fig. 4B). Therefore, our findings suggest that luteolin hinders DC maturation in the context of allotransplantation.

Foxp3 is specifically expressed in CD4⁺CD25⁺ Tregs that play a critical role in regulating immunity and maintaining immune homeostasis (27, 28). We further asked if luteolin would upregulate CD4⁺Foxp3⁺ Tregs because it ameliorated skin allograft rejection. Ten days after skin transplantation, draining LN and spleen cells were isolated from C57BL/6 mice, and CD4⁺Foxp3⁺ Tregs were enumerated by FASC analysis. As shown in Fig. 5A, the frequencies of CD4⁺Foxp3⁺ Tregs in draining LNs were significantly increased by luteolin treatment compared with the control without luteolin (p < 0.01). As expected, Rapa also increased their frequencies in LNs. However, there was no significant change in the frequencies of the splenic Tregs within all groups. We further analyzed Foxp3 expression in an allograft via immunofluorescence staining 10 d posttransplantation. We found that both Lut-low and Lut-high augmented Foxp3 expression in skin allografts, and so did Rapa (Fig. 5B). Thus, luteolin promoted CD4⁺Foxp3⁺ Treg generation in LNs as well as allografts in the context of allotransplantation.

Because luteolin increased CD4⁺Foxp3⁺ Treg frequency in vivo, we decided to examine whether luteolin would enhance the generation of CD4⁺Foxp3⁺ Tregs in vitro. CD4⁺CD25^– T cells were sorted from naive C57BL/6 mice and stimulated with anti-CD3 and anti-CD28 Abs plus rIL-2 in the absence or presence of Lut-low (2.5 μM) or Lut-high (5.0 μM) or Rapa (positive control) for 4 d. Then, frequencies of CD4⁺Foxp3⁺ Tregs were detected via FACS analysis. As shown in Fig. 6, we found that either Lut-low or Lut-high obviously induced CD4⁺Foxp3⁺ Tregs compared with control group (p < 0.01). These results indicate that luteolin can also promote CD4⁺Foxp3⁺ Treg generation in vitro.

Because we found that luteolin reduced effector T cell frequencies in vivo, we further determined whether it would also suppress T cell proliferation and effector cytokine secretion in vitro. FACS-sorted CD3⁺ T cells from naive C57BL/6 mice were stained with CFSE and stimulated with anti-CD3 and anti-CD28 Abs plus rIL-2 in the absence or presence of luteolin or Rapa for 4 d. We found that luteolin significantly suppressed T cell proliferation, and so did Rapa (Fig. 7A). Furthermore, we collected the supernatant to measure the protein levels of IFN-γ, IL-10, and IL-17a via ELISA. As shown in Fig. 7B, luteolin significantly lowered IFN-γ and IL-17a protein levels, whereas it promoted IL-10 secretion in the supernatant compared with the control group. In contrast, mTOR signaling is reportedly associated with the suppressive function of Tregs and Rapa is an mTOR inhibitor that can induce CD4⁺Foxp3⁺ Tregs (29). In the current study, we found that luteolin induced CD4⁺Foxp3⁺ Tregs in vivo and vitro. Thus, we examined whether luteolin would have an impact on mTOR signaling in T cells. The protein expressions of AKT, P-AKT, P-p70S6K, p70S6K, P-mTOR, and mTOR in CD3⁺ T cells were measured through Western blotting. As shown in Fig. 7C, luteolin effectively inhibited the expressions of P-p70S6K (at either low or high concentration: p < 0.01) as well as P-mTOR (p < 0.01) compared with control group, and so did Rapa. However, only Lut-high but not Lut-low reduced unphosphorylated mTOR expression. Importantly, luteolin, but not Rapa, suppressed AKT expression and its phosphorylation. At the concentrations used for in vitro suppression, luteolin was not cytotoxic, as determined by CCK-8 assays (Supplemental Fig. 1). These findings suggest that luteolin suppresses proliferation of T cells and their AKT/mTOR signaling but does not promote their death.

To determine if an increase in Tregs in luteolin-treated mice plays a major role in allograft survival extended by luteolin, B6 mice were transplanted with skin from BALB/c mice and treated with luteolin and depleting anti-CD25 Ab (PC61) or isotype Ab. As shown in Fig. 8A, Lut-high significantly prolonged skin allograft survival while depleting CD4⁺CD25⁺ Tregs largely, although not totally, reversed the luteolin-induced extension of skin allograft survival. However, isotype control for PC61 did not reverse the allograft survival. In contrast, we also measured the percentages of CD80/86⁺ mature DCs within CD11c⁺ cells 10 d after skin allotransplantation. We found that Lut-high significantly reduced the percentages of CD80/86⁺ mature DCs in both LNs and spleens (Fig. 8B). Depleting CD4⁺CD25⁺ Tregs also largely reversed the luteolin-induced downregulation of CD80/86⁺ mature CD11c⁺ DCs, suggesting that increased Tregs by luteolin may be responsible, at least in part, for luteolin-induced downregulation of mature DCs.

Immunosuppressive regimens are widely required for prevention of both acute and chronic transplantation rejection, but graft survival is still limited because of the toxicity and other side effects associated with global immunosuppression (6, 10). Newly emerging immunosuppressants are always under development to improve the longevity of transplanted organs and patients’ quality of life. Luteolin, a plant flavonoid derived from omnipresent herbal medicine or edible foods, has been shown to possess an anti-inflammatory feature (16). In this study, we used a mouse model of skin allotransplantation to determine an effect of luteolin on allograft rejection and to identify the potential mechanisms underlying its suppression of transplant rejection. Our results demonstrated that luteolin significantly prolonged skin allograft survival, ameliorated inflammatory cellular infiltration, and downregulated proinflammatory cytokine gene expression in a skin allograft. Further, it increased the frequency of CD4⁺Foxp3⁺ Tregs while restricting the expansion of mature DCs and effector CD4⁺/CD8⁺ T cells in vivo. Luteolin also suppressed T cell proliferation and induced CD4⁺Foxp3⁺Tregs in vitro. More importantly, luteolin significantly inhibited AKT/mTOR signaling in T cells in vitro. Therefore, luteolin is likely an emerging immunosuppressant as an mTOR inhibitor, although its safety/toxicity and potential clinical application remain unknown.

To determine an effect of luteolin on allograft rejection, we used BALB/c mice as donors and C57BL/6 mice as recipients for skin transplantation. We found that luteolin treatments significantly prolonged skin allograft survival. Infiltration with overall proinflammatory cells or CD3⁺ T cells was inhibited, whereas gene expressions of proinflammatory cytokine of IFN-γ, IL-6, IL-17a, and TNF-α were downregulated in skin allografts by luteolin treatment. Moreover, luteolin reduced the frequency of CD44^highCD62L^low effector CD4⁺/CD8⁺ T cells in vivo and suppressed T cell proliferation in vitro as well. T cells play a central role in the process of transplant rejection, and depletion or suppression of effector T cells is key to preventing rejection (1–3). Thus, it is possible that luteolin suppresses allograft rejection by direct inhibition of T cell activation. In contrast, DCs play a critical role in T cell activation, whereas CD11c-expressing DCs in recipients accelerate allograft rejection and expand alloreactive CD4⁺ and CD8⁺ T cells (30). We found that luteolin hindered DC maturation in both LNs and spleens posttransplantation. Thus, it is also possible that luteolin indirectly inhibits T cell activation and function by interfering with DC-mediated Ag presentation and recognition.

Tregs are critical in maintaining immune homeostasis and tolerance (31–33), although they are a small subset (5–10%) of CD4⁺ T cells. Foxp3 is specifically expressed in CD4⁺CD25⁺ Tregs and plays a critical role in regulating Treg development and function (27, 28). TGF-β1 is required to maintain Treg function and Foxp3 expression (34, 35). Induction of endogenous CD4⁺CD25⁺ Tregs or adoptive transfer of exogenous ones prevents autoimmune diseases and allograft rejection in many animal models (36–38). In this study, luteolin increased the frequencies of CD4⁺Foxp3⁺ Tregs in draining LNs and spleens of recipient mice. Further, we demonstrated that luteolin also augmented CD4⁺Foxp3⁺ Treg numbers in skin allografts while inducing the Tregs in vitro. Thus, our data indicate that the inhibitory effects of luteolin on allograft rejection and alloimmunity may be attributed to the increase in CD4⁺Foxp3⁺ Treg frequency.

We attempted to identify the molecular mechanisms underlying the effects of luteolin on allograft rejection. We found that luteolin treatment significantly reduced phosphorylation of mTOR and p70S6K as well as AKT. Rapa, a typical mTOR inhibitor that blocks mTORC1, is used to prevent allograft rejection. In recent years, mounting research has shown that Rapa not only suppresses T cell activation and proliferation, but also expands CD4⁺CD25⁺Foxp3⁺ Tregs (39, 40). The mTOR signaling pathway plays a pivotal role in regulating T cell activation and Treg differentiation (24, 41–43). Therefore, inhibition of mTOR activity is a critical therapeutic approach for antirejection treatments because it tips the balance between effector T cells and Tregs. Interestingly, we demonstrated that luteolin, but not Rapa, inhibited AKT expression and its phosphorylation, whereas it is well known that Rapa blocks mTORC1. Therefore, although both luteolin and Rapa are mTOR inhibitors, they target different molecules in the mTOR signaling pathway. Our data also indicate that luteolin is nearly as effective as Rapa in promoting CD4⁺Foxp3⁺ Treg generation and suppressing phosphorylation of p70S6K, a downstream mTOR pathway. More studies are needed to further characterize luteolin and its safety/toxicity.

Although we found that luteolin downregulated the frequency of effector T cells and mature DCs while inducing CD4⁺Foxp3⁺ Tregs, it remains to be defined which mechanism is more important for allograft survival extended by luteolin. Based on our findings, luteolin inhibits mTOR signaling pathway by targeting AKT in T cells and thus suppresses their proliferation while inducing Tregs. By targeting mTOR signaling, luteolin alters the balance between effector T cells and Tregs. These are likely main mechanisms underlying allograft survival extended by luteolin. It is well known that blocking mTOR signaling by Rapa promotes Treg generation and suppresses allograft rejection. Although both luteolin and Rapa suppress mTOR signaling in T cells, Rapa targets mTORC1, whereas we have demonstrated that luteolin acts on its upstream AKT. Furthermore, we found that Treg depletion largely reversed luteolin-mediated prolongation of allograft survival, indicating that upregulation of Tregs by luteolin may account for most of its effects on allograft survival. We also found that a reduction in percentage of CD80⁺/86⁺ within CD11c⁺ DCs by luteolin was mostly undone by Treg depletion, suggesting that upregulation of Tregs by luteolin, rather than its direct effects on DCs, may also be responsible, at least in part, for a decrease in mature DCs.

Taken together, a natural ingredient luteolin suppresses allograft rejection, accompanied with induction of CD4⁺Foxp3⁺ Tregs, reduction in effector T cell frequency, and inhibition of DC maturation. It also inhibits proliferation of T cells and their AKT/mTOR signaling. Thus, luteolin is an emerging immunosuppressant as a novel mTOR inhibitor.

The authors have no financial conflicts of interest.