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. 2017 Mar 23;8(3):e2695.
doi: 10.1038/cddis.2017.86.

The mTOR signal regulates myeloid-derived suppressor cells differentiation and immunosuppressive function in acute kidney injury

Chao Zhang  1   2 , Shuo Wang  1   2 , Jiawei Li  1 , Weitao Zhang  2 , Long Zheng  1 , Cheng Yang  1   2 , Tongyu Zhu  1   2   3 , Ruiming Rong  1   2   4
Affiliations

The mTOR signal regulates myeloid-derived suppressor cells differentiation and immunosuppressive function in acute kidney injury

Chao Zhang et al. Cell Death Dis. .

Abstract

The mammalian target of rapamycin (mTOR) signal controls innate and adaptive immune response in multiple immunoregulatory contexts. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells of potent immunosuppressive capacity. In this study, we aimed to investigate the role of MDSCs in the protection of acute kidney injury (AKI) and the regulation of mTOR signal on MDSC's protective role in this context. In mice AKI model, rapamycin administration was associated with improved renal function, restored histological damage and decreased CD4+ and CD8+ T-cell infiltration in kidney tissue. MDSCs, especially CD11b+Ly6G+Ly6Clow G-MDSCs were recruited to the injured kidney following the interaction of CXCL1, CXCL2 and their receptor CXCR2 after inhibiting mTOR signal with rapamycin treatment. The adoptive transfer of rapamycin-treated MDSCs into the mice with AKI significantly improved the renal function, ameliorated histologic damages and limited the infiltration of T cells in kidney tissue. In addition, the expression of pro-inflammatory cytokines IL-1β and IFN-γ mRNA was downregulated while the expression of TGF-β1 and Foxp3 mRNA was upregulated in kidney tissue after transferring rapamycin-treated MDSCs. Adoptive transfer of rapamycin-treated MDSCs also downregulated the serum levels of IL-1β, IL-6 and IFN-γ and upregulated the serum levels of TGF-β1 compared with the IR group and PBS-treated MDSC group. In in vitro study, inhibiting mTOR signal regulated the induction of MDSC towards the CD11b+Ly6G+Ly6Clow G-MDSC subset. The ability to suppress T-cell proliferation of both bone marrow-derived CD11b+Ly6G+Ly6Clow G-MDSCs and CD11b+Ly6G-Ly6Chigh M-MDSCs was enhanced by mTOR signal inhibition via upregulating the expression of Arginase-1 and iNOS. Accordingly, both G-MDSCs and M-MDSCs presented downregulated runx1 gene expression after rapamycin treatment. Taken together, our results demonstrated that MDSCs ameliorated AKI and the protective effect was enhanced by mTOR signal inhibition via promoting MDSCs recruitment, regulating the induction of MDSCs and strengthening their immunosuppressive activity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Rapamycin protects mice kidney against AKI in vivo. (a) The level of serum creatinine (μmol/l) and blood urea nitrogen (mmol/l) on POD 1 and POD 2. (b)The histological image of H&E-stained kidney tissue (× 200). Semi-quantitative analysis was performed following histological scoring system. (c) Immunohistochemical staining of CD4 and CD8 in kidney tissue (× 200). The black arrow pointed to the positive cells. The average number of positive cells per field was analyzed. (data were shown as mean±S.D.; n=5 mice per group per time point; POD, postoperative day)
Figure 2
Figure 2
MDSCs recruit to injured kidney following rapamycin treatment. CD11b+ cells were first gated from FSC/SSC, and then Ly-6G+Ly-6Clow and Ly-6G−Ly-6Chigh cell populations were detected within CD11b+ cells. (a) The frequency of CD11b+Ly-6G+Ly-6Clow G-MDSCs, CD11b+Ly-6G−Ly-6Chigh M-MDSCs and total MDSCs in spleen on POD 1 and POD 2 were examined by flow cytometry. (b) The frequency of CD11b+Ly-6G+Ly-6Clow G-MDSCs, CD11b+Ly-6G−Ly-6Chigh M-MDSCs and total MDSCs in the kidney on POD 1 and POD 2 were examined by flow cytometry. (data were shown as mean±S.D.; n=5 mice per group per time point; POD, postoperative day)
Figure 3
Figure 3
CXCL1, CXCL2 and CXCR2 interaction mediates the recruitment of MDSCs in injured kidney. (a) The expression of chemokine CXCL1, CXCL2 mRNA in kidney were upregulated on POD 1 and POD 2, but the expression of CXCL3 and CXCL7 mRNA were not (the expression was normalized to GAPDH and relative to control). (b) The percentage of CD11b+CXCR2+ cells in the kidney on POD 1 and POD 2 were increased in the rapamycin-treated group compared with sham group and the IR group. Cells in the kidney were gated first in FSC/SSC, and then CD11b+CXCR2+ cells were detected by flow cytometry. (data were shown as mean±S.D.; n=5 mice per group per time point; POD, postoperative day)
Figure 4
Figure 4
Adoptive transfer of MDSCs protects kidney against AKI and mTOR signal inhibition enhances MDSCs' protective effects. (a) The level of serum creatinine (μmol/l) and blood urea nitrogen (mmol/l) were decreased after adoptive transfer of MDSCs and further decreased after adoptive transfer of rapamycin-treated MDSCs. (b) Renal histologic damage was assessed in H&E-stained sections. Mild-to-moderate tubular vacuolation and hemorrhage were observed in the kidneys in the MDSC group compared with the IR group, and the histologic damage of kidneys in Rapa+MDSC group was even milder. (c) The percentage of apoptotic cells (shown as dark purple in color) in kidney tissues in situ was detected and quantified by using TUNEL assay. (d) Flow cytometry analysis showed the percentage of infiltrated CD4+ T cells in kidney tissues after adoptive transfer of non-rapamycin-treated MDSCs and rapamycin-treated MDSCs. (e) The expression of IL-1β, IL-6, IFN-γ, TGF-β1 and Foxp3 mRNA in the kidney was examined by RT-qPCR. (f) The serum level of cytokine IL-1β, IL-6, IFN-γ and TGF-β1 was measured by using Luminex and ELISA kit. (data were shown as mean±S.D.; n=5 mice per group; IHC, immunohistochemistry; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TUNEL, TdT-mediated dUTP Nick-End Labeling)
Figure 4
Figure 4
Adoptive transfer of MDSCs protects kidney against AKI and mTOR signal inhibition enhances MDSCs' protective effects. (a) The level of serum creatinine (μmol/l) and blood urea nitrogen (mmol/l) were decreased after adoptive transfer of MDSCs and further decreased after adoptive transfer of rapamycin-treated MDSCs. (b) Renal histologic damage was assessed in H&E-stained sections. Mild-to-moderate tubular vacuolation and hemorrhage were observed in the kidneys in the MDSC group compared with the IR group, and the histologic damage of kidneys in Rapa+MDSC group was even milder. (c) The percentage of apoptotic cells (shown as dark purple in color) in kidney tissues in situ was detected and quantified by using TUNEL assay. (d) Flow cytometry analysis showed the percentage of infiltrated CD4+ T cells in kidney tissues after adoptive transfer of non-rapamycin-treated MDSCs and rapamycin-treated MDSCs. (e) The expression of IL-1β, IL-6, IFN-γ, TGF-β1 and Foxp3 mRNA in the kidney was examined by RT-qPCR. (f) The serum level of cytokine IL-1β, IL-6, IFN-γ and TGF-β1 was measured by using Luminex and ELISA kit. (data were shown as mean±S.D.; n=5 mice per group; IHC, immunohistochemistry; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; TUNEL, TdT-mediated dUTP Nick-End Labeling)
Figure 5
Figure 5
mTOR signal regulates the induction of MDSCs from bone marrow cells. (a) CD11b+ cells were gated first, and Ly-6G+Ly-6Clow and Ly-6G−Ly-6Chigh cell populations were detected within CD11b+ cells. In comparison with control, rapamycin treatment directed MDSC induction towards Ly-6G+Ly-6Clow G-MDSC subtype. (b) After 100 nM rapamycin or equal volume of DMSO treatment for 4 h, the frequencies of CD4+IFN-γ+ Th1 cells, CD4+IL-17a+ Th17 cells and CD8+IFN-γ+ T cells were examined by using flow cytometry, respectively. (data were shown as mean±S.D.; n=5 duplicate tests)
Figure 6
Figure 6
mTOR inhibition with rapamycin promotes immunosuppressive activity of MDSCs in vitro. (a) CD11b+Ly-6G+Ly-6Clow G-MDSCs and CD11b+Ly-6G−Ly-6Chigh M-MDSCs with DMSO or rapamycin treatment were co-cultured with CFSE-stained CD4+ T cells at the ratio of 1:10 and 1:3 for 5 days, respectively, then the proliferation of T cells was assayed by flow cytometry. The decreased percentage of CFSE MFI after DMSO or rapamycin treatment indicated the ability of suppression of T-cell proliferation. Rapamycin treatment promoted immunosuppressive function of both M-MDSCs and G-MDSCs compared with control. (b) Arginase-1 and iNOS mRNA expression in CD11b+Ly-6G−Ly-6Chigh M-MDSCs and CD11b+Ly-6G+Ly-6Clow G-MDSCs were examined by RT-PCR. The expression of Arginase-1 and iNOS mRNA were upregulated in M-MDSCs, whereas in G-MDSCs only Arginase-1 mRNA was upregulated after rapamycin treatment. (c) Arginase-1 and iNOS protein levels in CD11b+Ly-6G+Ly-6Clow G-MDSCs and CD11b+Ly-6G−Ly-6Chigh M-MDSCs were examined by western blot. For both G-MDSCs and M-MDSCs, the protein levels of Arginase-1 and iNOS were increased after rapamycin treatment when compared with the control group. (d)The protein levels of Runx1 in G-MDSCs and M-MDSCs with or without rapamycin treatment were examined by western blot. The levels of Runx1 protein in both G-MDSCs and M-MDSCs were significantly reduced after rapamycin treatment in comparison with the control groups. There was almost no Runx1 expression in G-MDSCs after rapamycin treatment. (e) The levels of runx1 mRNA in G-MDSCs and M-MDSCs were examined by RT-PCR. Both G-MDSCs and M-MDSCs presented downregulated expression of runx1 mRNA after rapamycin treatment, however, the decrease of runx1 mRNA in M-MDSCs was not significant. (data were shown as mean±S.D.; n=5 duplicate tests; Arg-1, Arginase-1; CFSE, carboxyfluorescein succinimidylester; DMSO, dimethyl sulfoxide; MFI, mean fluorescence intensity)
Figure 7
Figure 7
The schematic figure explicating the regulation of mTOR signal on myeloid-derived suppressor cells in acute kidney injury. Inhibition of mTOR signal with rapamycin enables to enhance the protective role of MDSCs via promoting MDSCs recruitment to injured kidney, regulating the induction of MDSCs from bone marrow cells and strengthening its immunosuppressive activity
See this image and copyright information in PMC

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References

    1. Bellomo R, Kellum JA, Ronco C. Acute kidney injury. Lancet 2012; 380: 756–766. - PubMed
    1. Mehta RL, Cerda J, Burdmann EA, Tonelli M, Garcia-Garcia G, Jha V et al. International Society of Nephrology's 0by25 initiative for acute kidney injury (zero preventable deaths by 2025): a human rights case for nephrology. Lancet 2015; 385: 2616–2643. - PubMed
    1. Doi K, Rabb H. Impact of acute kidney injury on distant organ function: recent findings and potential therapeutic targets. Kidney Int 2016; 89: 555–564. - PubMed
    1. Rabb H, Daniels F, O'Donnell M, Haq M, Saba SR, Keane W et al. Pathophysiological role of T lymphocytes in renal ischemia-reperfusion injury in mice. Am J Physiol Renal Physiol 2000; 279: F525–F531. - PubMed
    1. Burne-Taney MJ, Yokota N, Rabb H. Persistent renal and extrarenal immune changes after severe ischemic injury. Kidney Int 2005; 67: 1002–1009. - PubMed

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