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. 2017 Oct 11;8(53):90766-90780.
doi: 10.18632/oncotarget.21798. eCollection 2017 Oct 31.

The TRPV1 ion channel regulates thymocyte differentiation by modulating autophagy and proteasome activity

Consuelo Amantini  1 , Valerio Farfariello  2 , Claudio Cardinali  3   4 , Maria Beatrice Morelli  3   4 , Oliviero Marinelli  1 , Massimo Nabissi  3 , Matteo Santoni  3 , Laura Bonfili  1 , Valentina Cecarini  1 , Anna Maria Eleuteri  1 , Giorgio Santoni  3
Affiliations

The TRPV1 ion channel regulates thymocyte differentiation by modulating autophagy and proteasome activity

Consuelo Amantini et al. Oncotarget. .

Abstract

Autophagy and the ubiquitin-proteasome system (UPS) control thymus cell homeostasis under resting and endoplasmic reticulum (ER) stress conditions. Several evidence support a cross-talk between UPS and autophagy; abrogation of UPS responses stimulates autophagy, and vice versa the inhibition of autophagy alters the UPS functions. Herein, we found that TRPV1 activation induces ER stress, proteasome dysfunction and autophagy in thymocytes by modulating the expression of UPR-related genes. The TRPV1-mediated autophagy prevents the UPR activation by inhibiting BiP, Grp94 and ERp57 chaperone protein expression. Thymocytes from TRPV1 KO mice display both autophagy and proteasome dysfunctions, resulting in increased apoptotic cells and reduced total DP thymocyte number. In addition, positive selection of thymocytes triggered by anti-TCRβ/CD2 Ab-mediated costimulation induces apoptosis in thymocytes from TRPV1 KO as compared with WT mice. Stimulation of TRPV1 KO thymocytes with anti-TCRβ/CD2 mAbs modulates the expression of CD4 antigen on purified DP thymocytes, with reduced number of mature, single positive (SP) CD4 and increased number of immature SP CD4low and DP CD4lowCD8+ thymocytes, further supporting the intrinsic role of TRPV1 in T cell maturation. Finally, a reduction in CD8+ and CD4+ T cells is evidenced in the peripheral blood and spleen of TRPV1 KO, as compared with WT mice. Therapeutic strategy by restraining or stimulating the TRPV1 expression and functions in thymocytes might represent a new pharmacological tool in the regulation of different inflammatory T cell responses.

Keywords: ER stress; Immune response; Immunity; Immunology and Microbiology Section; TRPV1; TRPV1 KO mice; autophagy; capsaicin.

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Conflict of interest statement

CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1. CPS inhibits cellular proteasome activity
A. WT thymocytes were subjected to enzymatic proteasome activity assay after 30, 60 and 120 minutes of CPS treatment. Data, shown as percentage of proteasome activity, are from one representative experiment out of three separate experiments. Error bars are relative to three replicates. *p < 0.01 vs untreated cells. B. Western blot analysis and densitometric quantification of p27 protein levels in thymocytes treated for up to 120 minutes with CPS. p27 densitometry values were normalized to GAPDH used as loading control. Blots are representative of one of three separate experiments. *p < 0.01 treated vs untreated cells.
Figure 2
Figure 2. CPS-mediated inhibition of enzymatic proteasome activity is ROS-, Ca2+- and TRPV1- dependent
A. Enzymatic proteasome activities were assessed in WT thymocytes pre-treated with NAC, EDTA and CPZ for 60 minutes and then treated with CPS for 120 minutes. Data, shown as percentage of proteasome activity, are from one representative experiment out of three separate experiments. Error bars are relative to three replicates. *p < 0.01 vs untreated cells; **p < 0.01 vs CPS-treated cells. B. Western blot analysis and densitometric quantification of p27 protein levels in thymocytes treated as above described. p27 densitometric values were normalized to GAPDH used as loading control. Blots are representative of one of three separate experiments. #p < 0.01 vs untreated; **p < 0.01 vs CPS-treated cells.
Figure 3
Figure 3. The 4-PBA abrogates the CPS-induced autophagy and reduces the DPdull thymocyte subpopulation
A. Western blot analysis of LC3 and p62 protein in WT thymocytes pre-treated with 4-PBA for 60 minutes and then treated for 120 minutes with CPS. The ratio of LC3 II/I was calculated from densitometric data. p62 densitometric values were normalized to GAPDH used as loading control. Blots are representative of one of three separate experiments. *p < 0.01 vs untreated cells, **p < 0.01 vs CPS-treated cells. B. Flow cytometric analysis was performed by Annexin V-FITC and PI double-staining of WT thymocytes, pre-treated with 4-PBA for 60 minutes and then treated for 120 minutes with CPS. Data represent the percentage of PI and/or Annexin V positive cells. One representative out of three independent experiments is shown. C. Thymocytes, pre-treated with 4-PBA, CQ or NAC for 60 minutes and then treated for 120 minutes with CPS, were stained with anti-CD4-PE and anti-CD8-Cy5 mAbs and analyzed by FACS. The black gate indicates the DPdull subpopulation. One representative out of three independent experiments is shown.
Figure 4
Figure 4. CPS reduces UPR protein levels, and autophagy reverts CPS-induced inhibitory effects
A. Western blot analysis and densitometric quantification of Grp94, BiP and ERp57 protein levels in WT thymocytes pre-treated with CQ and 4PBA for 1h and then treated for up to 4h with CPS. Blots are representative of one of three separate experiments. B. Densitometric quantification of Grp94, BiP and ERp57 protein levels in WT thymocytes treated with CPS for different times. Densitometric values were normalized to GAPDH used as loading control and represent the mean ± SEM of three separate experiments. *p < 0.01 vs untreated cells. C. Densitometric quantification of Grp94, BiP and ERp57 protein levels in thymocytes pre-treated with CQ and 4PBA and then treated with CPS. *p < 0.01 vs CPS-treated cells. Densitometric values were normalized to GAPDH used as loading control and represent the mean ± SEM of three separate experiments.
Figure 5
Figure 5. Knock-out of TRPV1 gene affects autophagy, proteasome and UPR protein expression in thymocytes
A. Western blot analysis and densitometric quantification of basal p62 protein levels in WT and TRPV1 KO thymocytes. Densitometric values were normalized to GAPDH used as loading control. Blots are representative of one of three separate experiments, *p < 0.01 vs WT thymocytes. B. Western blot analysis and densitometric quantification of LC3 and p62 protein levels in TRPV1 KO thymocytes treated for up to 4 h with rapamycin. Densitometric values were normalized to GAPDH used as loading control. Blots are representative of one of three separate experiments, *p < 0.01 vs CPS-treated cells for 0.5h or untreated cells. C. Thymocytes from WT and TRPV1 KO mice were subjected to enzymatic proteasome activity assay. Data shown as percentage of inhibition are representative of one of three separate experiments, *p < 0.01 TRPV1 KO vs WT thymocytes. D-I. Western blot analysis and densitometric quantification of p27 D., pAMPK/AMPK E., ATF-4 F., ERp57 G., Bip H. and Grp94 I. protein levels in WT and TRPV1 KO thymocytes. Densitometric values were normalized to GAPDH used as loading control. Blots are representative of one of three separate experiments, *p < 0.01 vs WT thymocytes.
Figure 6
Figure 6. Increased basal levels of apoptotic thymocytes and reduced total thymocyte number in TRPV1 KO mice
Freshly purified thymocytes from WT and KO mice were counted using trypan blue solution A., or stained with Annexin-V FITC and analyzed by FACS analysis B. Data are the mean ± SEM of three separate experiments, *p < 0.01 vs WT thymocytes. C. From the total number of cells per thymus, the relative number of cells in each CD4/CD8 subset was determined staining TRPV1 KO and WT thymocytes with anti-CD4-PE and anti-CD8-Cy5 mAbs and using FACS analysis. Data are the mean ± SEM of three separate experiments *p < 0.01 TRPV1 KO vs WT thymocytes.
Figure 7
Figure 7. Knock-out of TRPV1 gene impairs positive thymic selection and affects peripheral blood and spleen CD4+ and CD8+ T cell numbers
A. Thymocytes from WT and KO mice, stimulated with anti-TCRβ plus anti-CD2 mAbs after 18h of culture and 12h of recovery, were analyzed by FACS analysis. One representative out of three independent experiments is shown. B. Purified DP thymocytes from TRPV1 KO and WT mice stimulated with TCRβ plus CD2 mAbs for 18h, followed by 12h recovery, were stained with anti-CD4-PE and anti-CD8-Cy5 mAbs and analyzed by FACS. One representative out of three independent experiments is shown. C. Blood and spleen PBMC from WT and KO thymocytes were stained with anti-CD4-PE and anti-CD8-Cy5 mAbs and analyzed by FACS. One representative out of three independent experiments is shown in each panel.
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