IL‐27 Rα+ cells promoted allorejection via enhancing STAT1/3/5 phosphorylation

Abstract Recently, emerging evidence strongly suggested that the activation of interleukin‐27 Receptor α (IL‐27Rα) could modulate different inflammatory diseases. However, whether IL‐27Rα affects allotransplantation rejection is not fully understood. Here, we investigated the role of IL‐27Rα on allorejection both in vivo and in vitro. The skin allotransplantation mice models were established, and the dynamic IL‐27Rα/IL‐27 expression was detected, and IL‐27Rα+ spleen cells adoptive transfer was performed. STAT1/3/5 phosphorylation, proliferation and apoptosis were investigated in mixed lymphocyte reaction (MLR) with recombinant IL‐27 (rIL‐27) stimulation. Finally, IFN‐γ/ IL‐10 in graft/serum from model mice was detected. Results showed higher IL‐27Rα/IL‐27 expression in allografted group compared that syngrafted group on day 10 (top point of allorejection). IL‐27Rα+ spleen cells accelerated allograft rejection in vivo. rIL‐27 significantly promoted proliferation, inhibited apoptosis and increased STAT1/3/5 phosphorylation of alloreactive splenocytes, and these effects of rIL‐27 could be almost totally blocked by JAK/ STAT inhibitor and anti‐IL‐27 p28 Ab. Finally, higher IL‐27Rα+IFN‐γ+ cells and lower IL‐27Rα+IL‐10+ cells within allografts, and high IFN‐γ/low IL‐10 in serum of allorejecting mice were detected. In conclusion, these data suggested that IL‐27Rα+ cells apparently promoted allograft rejection through enhancing alloreactive proliferation, inhibiting apoptosis and up‐regulating IFN‐γ via enhancing STAT pathway. Blocking IL‐27 pathway may favour to prevent allorejection, and IL‐27Rα may be as a high selective molecule for targeting diagnosis and therapy for allotransplantation rejection.

further understanding rejection mechanisms and modulating clinic administration.

Interleukin-27 receptor (IL-27R) is composed of WSX-1 (IL-27Rα)
and gp130 (signal-transducing subunit), expressed on CD4 + T cells, activated macrophages and neutrophils. 4 And IL-27, the ligand of IL-27Rα, belongs to IL-6/IL-12 family. 5 It was reported that engraftment of IL-6 deficient donor into wild-type recipient could significantly improve allograft survival through T cell lineage particularly regulatory T cells (Tregs) in non-sensitized transplant host. 6 IL-27 could directly promote Th1 cell response, meanwhile inhibit the development and effector response of Th17 cells. 7 And more importantly, data about IL-27 displayed a controversial effect on transplantation rejection.
Ayasoufi K proved that IL-27 neutralization or receptor deficiency delayed CD8 + T cell recovery after murine anti-thymocyte globulin (mATG) treatment in heart allotransplantation mouse model, 8  increasing CD4 + IL-10 + IFN-γ + T cells production, 10 up-regulation of IL-27 combined with rapamycin promoted IL-10 + CD4 + T cells effect in prolonging cardiac allograft survival, 11 which suggested that IL-27 and IL-27Rα inhibited allorejection. So, the effect of IL-27Rα/IL-27 on allotransplantation rejection and its underlying mechanism was not fully understood yet.
Our previously study proved that IL-27Rα up-regulated 4 folds in allogeneic CD4 + T cells, and more importantly, we found 10-fold up-regulation of Stat1 and 4 folds increasing of interferon gamma receptor 1 (Ifngr1) at the same kind of CD4 + T cells, compared with syngeneic CD4 + T cells in Serial analysis gene expression (SAGE).
Considering of the key role of CD4 + T cells in allorejection, and the enhancing effect of IFN-γ on allograft rejection, 12 we postulated that IL-27Rα, the upstream regulator of STAT1, may be the important target molecule on allograft rejection. Here, we investigated the effect of IL-27Rα during allograft rejection, and possible signal pathway, to clarify the effect of IL-27Rα as a pivotal molecule, on allotransplantation rejection.

| Animal models
Female BALB/c mice (H-2 d ), C57BL/6 mice (H-2 b ) and SCID Beige mice (BALB/c background) aged 6-8 weeks and weighted 18 ± 2 g were purchased from Vital River Laboratory Animal Technology and fed in SPF condition. The skin transplantation was performed according to reference. 13 BALB/c mice were recipients, and BALB/c and C57BL/6 were donors for syngeneic and allogeneic grafted group, respectively. The vaseline gauzes and bandage were packed on day 1 and removed on day 7 post-transplantation. Graft was observed daily. Rejection was determined when scab area of graft was more than 50%. Allografted SCID mice model was established according to reference. 14 The cell adoptive transfer for allo-SCID mice was performed after graft healed totally, then, 1.5*10 6 sorted IL-27Rα + cells and control non-sorted spleen cells isolated from allografted BALB/c mouse model on day 10 were adoptively transferred to SCID mouse model by intraperitoneal injection. Graft was observed daily. Rejection was determined when scab area of graft was more than 50%.
All animal studies were conducted in accordance with protocols approved by the Animal Care and Use Committee of Shandong University.

| Spleen cell isolation, culture and reagents treatment
Spleen was isolated from mice, and single cell suspension was prepared after treated with red blood cell lysis buffer (Solarbio) according to the manufacturer's instruction. The donor cells were treated with 40 μg/mL mitomycin C (Solarbio) in 37°C, 5% CO 2 for 30 minutes in dark and then washed three times with PBS. All cells were cultured in complete RPMI 1640 (Biological Industries) supplemented with 10% foetal bovine serum (FBS) (Biological Industries), and 100 U/mL Penicillin, and 100 µg/mL streptomycin (Hyclone) and cultured in 37°C, 5% CO 2 . The splenocytes were adjusted to a final concentration of 4 × 10 5 /well (recipient) and 2 × 10 5 /well (donor) in 96-well and 1 × 10 6 /well (recipient) and 5 × The final concentration of anti-IL-27 p28 Ab (R&D) is 7.5 µg/mL (dissolved in PBS). Cell counting kit-8 (CCK-8) (DOJINDO) reagent was added in cell suspension for 10 µL/well, and microplate reader was used to measure OD value in 490 nm to detect cells proliferation.

| Flow cytometry and cells sorting
Spleen cell suspension was prepared described as above, and cell number was adjusted into 1 ×

| H&E (haematoxylin-eosin) staining
Graft was separated and fixed overnight, then dehydrated and embedded in paraffin blocks. The tissue sections were deparaffinized, rehydrated and stained with H&E staining kit (Servicebio).
Briefly, the tissue sections were coated with haematoxylin for 5 minutes and washed with water, then covered with 1% acid ethanol regent for 5 seconds and washed with water, added bluepromoting solution for 5 seconds, then washed with water and covered with eosin solution for 10 minutes, and dehydrated with alcohol and clearing in xylene. The image was obtained under the optical microscope.

| Immunofluorescence and immunohistochemical staining
The graft tissue sections were deparaffinized, rehydrated and then antigen repaired with EDTA antigen repair buffer (pH 9.0) and

| Western blot
The tissue was smashed and cells were washed with ice-cold PBS and lyzed using radioimmunoprecipitation assay (RIPA) lysis buffer The membranes were washed three times with TBST buffer and incubated with secondary antibody (1:10 000, dissolved to TBST) for 1 hour at room temperature, then, washed three times with washing buffer and added HRP substrate peroxide solution (Millipore). The image was obtained by Tanon 5200 imaging system scanner and analysed by ImageJ software.

| ELISA (enzyme-linked immunosorbent assay)
IFN-γ, IL-27 and IL-10 in serum of mouse model were measured by ELISA Kit (Shanghai Enzyme-linked Biotechnology) following the manufacture' description. Briefly, the diluted sample was coated with conjugate reagent in the bottom of plate for 60 minutes at 37°C. The liquid was removed, and plate was washed with washing buffer for five times. Add the substrate solution for 15 minutes and then stop the colour reaction. The absorbance was measured by ELISA plate reader at OD 450 nm (Bio-Rad). Standard curve was established to quantity the concentration of the sample.

| Statistical analysis
Statistical analyses were performed using the GraphPad Prism 8 software and evaluated by two-tailed t tests in comparisons of two groups. The survival analysis was evaluated by comparison of survival curves. P values < .05 were considered to be statistically significant. The quantitative values of all experiments were presented as the mean ± SD and were calculated from at least 3 independent experiments.

| The dynamic expression of IL-27Rα/IL-27 in grafts post-transplantation
To investigate whether IL-27Rα/IL-27 expression in graft was correlated with allograft rejection, we established allogeneic skin grafted mouse model and with syngeneic skin grafted mouse model as control. Grafts were isolated on 1, 7, 10, 14 and 21 day post-transplantation, and IL-27Rα and IL-27 expression were detected, respectively. H&E staining showed whole process of allograft rejection from day 1 to 21 post-transplantation ( Figure 1A), much more inflammatory infiltration within graft, while no inflammation observed in syngeneic grafted group. Figure 1B,C showed that allograft rejection occurred on day 10, average survival time of allograft was 11 days, no rejection was observed in syngeneic grafted group. IHC staining showed high IL-27Rα expression on day 1 both in allograft and syngraft, the expression of IL-27Rα was obviously increased from day 7, significantly high expressed on day 10 in allograft than syngraft (P < .05).
On day 21, accompanied with the inflammation decreased, lower IL-27Rα expression was detected both in two groups. IL-27Rα positive staining mostly was detected in connective tissue and newborn blood vessels within allograft ( Figure 1D

| IL-27Rα + cells promoted allograft rejection
Our previous study demonstrated IL-27Rα + T cells and IL-27Rα + CD68 + cells (macrophage) infiltrated in the graft. 15 Considering of the pivotal role of T cells in allorejection, 16 Immunofluorescence co-staining results showed as Figure 2A

| rIL-27 in vitro enhanced proliferation of alloreactive splenocytes via activating STAT1/3/5
Since the IL-27Rα + cells from spleen could promote allograft rejection, therefore, we further investigated the effective mechanism

| rIL-27 inhibited apoptosis of alloreactive cells via enhancing STAT1/3/5
Our previously data indicated that apoptosis of alloreactive cells played an important role in allorejection (Data not shown). According to report, 24 the downstream of STAT pathway includes cells proliferation and apoptosis. Considering that we already proved that proliferation increased by rIL-27 stimulation was through STAT pathway, further we investigated whether cell apoptosis was also affected through STAT pathway under rIL-27 stimulation. The results were shown as Figure 5A

| IL-27Rα activation was prone to higher IFN-γ and lower IL-10 expression on allorejection
Considering that pro-inflammatory/immunosuppressive cytokine both participated in alloreactive cells mediated rejection, 28 we  Figure 6A revealed the IL-27Rα + cells were up-regulation in allograft with higher IFN-γ and lower IL-10 expression simultaneously on day 10 post-transplantation. Compared with syngraft, the proportion of IL-27Rα + IL-10 + cells in allograft had a markedly reduction, while IL-27Rα + IFN-γ + cells obviously increased (P < .05, Figure 6B).
These results indicated that IFN-γ high expression and IL-10 low expression was affected on allorejection. Quantified with IOD/area value in Figure 6C further proved this result.
To understand the systemic effect of IL-27Rα high expression on allograft rejection, dynamic level of IFN-γ and IL-10 in serum of grafted mouse model post-transplantation were measured by ELISA.
The results showed that during transplantation rejection, the level of these cytokine changed depended on graft condition. Compared with the syngeneic grafted group, the allografted group showed F I G U R E 6 IFN-γ, IL-10 and IL-27 expression assay. Syngeneic and allogeneic skin mouse model was established and was represented by Syn and Allo group. IFN-γ, IL-10 and IL-27 level were measured in graft and serum. A, The IFN-γ (green), IL-10 (red) and IL-27Rα (pink) expression of graft was measured by immunofluorescent staining on day 10. The cell nucleus was stained with DAPI (blue). Scale bar = 20 μm. The arrow showed the IFN-γ + / IL-10 + and IL-27 Rα + cells. B, The per cent of IFN-γ or IL-10 positive cell in IL-27Rα positive cells on day 10. C, The IFN-γ or IL-10 expression was quantified by IOD/Area value. D, The level of IFN-γ, IL-10 and IL-27 in serum was measured by ELISA from day 1 to day 21 post-transplantation. *P < .05, **P < .01, ***P < .001 vs Syn group much higher IFN-γ and lower IL-10 on day 10 (P < .05, Figure 6D), which suggested that IL-27Rα high expression in grafts was positively correlated with higher pro-inflammatory and lower immunosuppressive cytokine in serum.
To investigate whether IL-27 in peripheral blood was also changed during allotransplantation rejection, IL-27 in model mice serum was also detected with ELISA, and the results showed that IL-27 also apparently increased on the day 10 after allogeneic transplantation (P < .05, vs syngeneic grafted group, Figure 6D), which confirmed that higher IL-27 was consisted with high expression of IL-27Rα and IL-27 within allograft when rejection occurred. Taken together, these results revealed that the IL-27Rα activation promoted much more IFN-γ and less IL-10 releasing, and these cytokine may be as an effective factor through STAT pathway to increase alloreactive cells proliferation, inhibit apoptosis, and to promote allografts allorejection.  34 IL-27 also strongly stimulated CD4 + T cell proliferation and Th1 cytokine production by up-regulating the antigen-processing capability of DC in the Staphylococcus aureus infection. 35 Considering that severe CD4 + T cell infiltration in graft and its key role in allorejection, 17  Recently, it was reported that IL-2Rα/IL-27 pathway displayed contrast effect in allogeneic transplantation rejection In humanized xenogeneic GVHD NOD/SCID model, IL-27 favoured human placenta-derived MSCs to promote generation of CD4 + IL-10 + IFN-γ + T cells and alleviate GVHD symptoms. 10 By enhancing IL-10/ IFN-γ expression ratio in the alloantigen specific CD4 + T cells, IL-27 adenovirus associated virus (AAV) transduction promoted the regulation of rapamycin to prolong the allograft survival. 11 In contrast, our present study showed that IL-27Rα was up-regulated in allogeneic CD4 + T cells. Recently report revealed that anti-IL-27 p28 antibody treatment could magnify the protection capability of TLR7 ligand R848 to GVHD by strongly activating regulatory T cell. 43 In our study, we found that anti-IL-27 p28 could almost totally neutralize that effect of rIL-27 on proliferation and apoptosis via STAT pathway.

| D ISCUSS I ON
In a summary, our data suggested that high expressed IL-27Rα in allogeneic graft promoted allorejection through enhancing proliferation and anti-apoptosis, via activating STAT pathway. The effective route may be mainly through producing much more pro-inflammatory cytokine IFN-γ and less immunosuppressive cytokine IL-10 in graft and in peripheral blood. Blocking IL-27 pathway may favour to prevent allorejection, and IL-27Rα may be as a high selective molecule for targeting diagnosis and therapy for allotransplantation rejection.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science Foundation of China (81371601, to Guihua Hou.) and Natural Science Foundation of Shandong Province (ZR2019MH019 to G. Hou).

CO N FLI C T S O F I NTE R E S T
The authors have no conflicts of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon request.