The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4⁺ T cells

Abstract

TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

Figure 1

TRPV1 is expressed in CD4⁺ T cells. (a) Splenic (SP) CD4⁺ T cells were isolated from C57BL/6 mice (i.e., WT) and expression of several Trp channels was analyzed by qPCR. Expression of Trp transcripts was normalized to Gapdh housekeeping gene and expressed relative to Trpm4 (known to be expressed in CD4⁺ T cells). Mean ± SEM of 5 mice are shown. n.s: not significant; *P <0.05; ***P <0.001; ****P <0.0001 (one-way ANOVA with post hoc Dunnett's test). (b) SP CD4⁺ T cells were isolated from WT and Trpv1^−/− mice and FACS-sorted CD4⁺TCRβ⁺ T cells (purity > 98%) were used to analyze TRPV1 expression by qPCR. PCR products from the qPCR reaction were separated on a 2% agarose gel. Trpv1-specific PCR product was detected with an expected size of 188 bp. Cd3d was used as a T cell marker and Gapdh as a loading control. (c) Splenocytes were isolated from WT mice, stained for CD4, TCRβ and TRPV1 and analyzed by flow cytometry. The histogram of TRPV1 expression on gated CD4⁺TCRβ⁺ T cells is shown (red line peak). The specificity of the TRPV1 Ab was confirmed by pre-incubating it with the corresponding blocking peptide (orange line peak). IgG control (grey peak). Geometric Mean Fluorescence (GMF) intensity is indicated. (d) Confocal images showing TRPV1 and CD4 subcellular localization in SP CD4⁺ T cells. DAPI (left panel), TRPV1-AF546 (mid-left panel), CD4-AF488 (mid-right panel) and the merge (right panel) are shown. Scale bar = 5 μm. Yellow color in the merge panel indicates high TRPV1 and CD4 colocalization. (e) TRPV1 and CD4 colocalization scatter plot was generated using Velocity®. Data are representative of three or more independent experiments.

Figure 2

TRPV1 is a functional in Ca²⁺ channel in CD4⁺ T cells. (a) Sample traces of inward currents recorded on WT, Trpv1^−/− and SB366791 (SB)-treated WT CD4⁺ T cells following application of the prototypical TRPV1 agonist, capsaicin (CAP, 16 μM). Cells were held at −85 mV. Horizontal bars denote the duration of the CAP exposure. Dotted lines indicate the zero current level. (b) Current density comparison between WT (n = 9) and Trpv1^−/− (n = 8) CD4⁺ T cells following CAP application. Values were obtained by normalizing peak current recorded at −85 mV to capacitance of each cell. Error bars represent mean ± SEM. **P <0.01 (two-tailed Student t-test). (c) Current density comparison between untreated (n = 10) and SB-treated (1 μM; n = 13) WT CD4⁺ T cells in response to CAP. Error bars represent mean ± SEM. ****p <0.0001 (two-tailed Student t-test). (d) Current-voltage relationship (I-V curve) of CAP-evoked current in CD4⁺ T cells exhibits outward rectification. A voltage ramp was delivered from −70 mV to +70 mV in 400-ms. CAP-evoked current was isolated by subtracting current before and after addition of CAP (3 μM). Currents were normalized relative to the current at −70 mV (−19.2±3.3 pA) and data is presented as the average of I-V curves from n = 4 cells. (e) WT (blue line), Trpv1^−/− (magenta line) and Trpv1^Tg (turquoise line) SP CD4⁺CD25⁻(naive) T cells were isolated, loaded with Fura-2 AM and changes in [Ca²⁺]_i following application of CAP (10 μM) in the presence of 2 mM CaCl₂ (2 Ca) were monitored by confocal imaging. (f) Statistical analysis of the Ca²⁺ influx profiles shown in e for CAP (1 or 10 μM). Mean ± SEM of 50-100 individual cells. *P <0.05; ***P <0.001; ****P <0.0001 (one-way ANOVA with post hoc Bonferroni test). (g) SP CD4⁺ T cells were isolated from WT and Trpv1^Tg mice and TRPV1 expression in total cell lysates was analyzed by immunoblotting. Immunoreactive doublets at ≈ 95 and 115 kDa correspond to the non-glycosylated and glycosylated forms of the TRPV1 channel. Data are representative of three or more independent experiments.

Figure 3

TRPV1^CD4 acts as a non-store-operated Ca²⁺ channel and contributes to TCR-induced Ca²⁺ influx. WT (blue line), Trpv1^−/− (magenta line) and Trpv1^Tg (turquoise line) SP CD4⁺CD25⁻ (naive) T cells were isolated, loaded with Fura-2 AM and changes in [Ca²⁺]_i were monitored by confocal imaging. (a) Cells were stimulated by anti-CD3 crosslinking in the presence of 2 mM CaCl₂ (2 Ca) and ionomycin (Iono, 1 μM) was added at the end of the acquisition. (b) Statistical analysis of the Ca²⁺ influx profiles shown in a. Mean ± SEM of 50-100 individual cells. (c) WT and Trpv1^−/− SP CD4⁺ T cells were isolated, loaded with Indo-1 AM and stimulated with soluble anti-CD3 (10 μg/mL) and anti-CD28 (1 μg/mL) in Ca²⁺-free medium. Changes in [Ca²⁺]_i were monitored by flow cytometry and overlay of the Ca²⁺ influx profiles after addition of CaCl₂ (2 mM/WT, 3 mM/Trpv1^−/−) to the extracellular medium is shown. (d) Store-operated Ca²⁺ measurements were performed after passive depletion of intracellular stores using 1 μM thapsigargin (TG). (e) Statistical analysis of the Ca²⁺ influx profiles shown in d. Mean ± SEM of 50-100 individual cells is shown. (f) Sample traces of inward currents in WT and Trpv1^−/− CD4⁺ T cells activated by anti-CD3 crosslinking and held at −85mV. Bar donates the duration of streptavidin application. (g) Comparison of TCR-induced currents in WT (n = 7) and Trpv1^−/− (n = 9) CD4⁺ T cells. Values were obtained by normalizing peak current to capacitance of each cell. (h) WT CD4⁺ T cells were pretreated with the indicated concentrations of a specific TRPV1 antagonist (BCTC) or the vehicle (VEH; 0.1% DMSO) for 5 min. Cells were then stimulated with anti-CD3+28 in Ca²⁺-free medium and CaCl₂ (5 mM) was added to the extracellular medium during the acquisition. (i) Statistical analysis of the Ca²⁺ influx profiles shown in f. Mean ± SEM of thrndent experiments is shown. n.s: not significant; *P <0.05; **P <0.01; ****p <0.0001 (two-tailed Student t-test [g], one-way ANOVA with post hoc Bonferroni test [b,e,i]). Data are representative of three or more independent experiments.

Figure 4

TRPV1^CD4 participates in TCR signaling. (a) Confocal images of Lck and TRPV1 clustering induced by anti-CD3 crosslinking. TRPV1 and Lck fluorescence signals largely colocalized in resting WT SP CD4⁺ T cells as indicated by the yellow color in the merge panel. In addition, TRPV1 is recruited to TCR clusters upon TCR liagtion (white arrowheads). The Src-family kinase inhibitor PP2 (10 μM) inhibits Lck and TRPV1 capping. Scale bar = 2 μm. (b) Jurkat cell clones E6.1 (WT) and J.Cam1.6 (Lck^−/−) were left unstimulated or stimulated with soluble anti-hCD3 (2.5 μg/mL) and anti-hCD28 (1 μg/mL) for 5 min and anti-TRPV1 immunoprecipitates (IP) or total cell lysates were analyzed by immunoblot (IB) with anti-phospho-tyrosine (p-Tyr) or with anti-TRPV1 and anti-Lck, respectively. (c) WT and Lck^−/− Jurkat T cells were loaded with Fura-2 AM and changes in [Ca²⁺]_i upon application of capsaicin (CAP, 1 μM) were monitored by confocal imaging. Ionomycin (Iono, 1 μM) was used as a positive control and was added at the end of the acquisition. Right panel: statistical analysis of the CAP-induced Ca²⁺ influx peak. n.s: not significant; ****P <0.0001 (two-tailed Student t-test). (d) WT and Trpv1^−/− SP CD4⁺ T cells were isolated and stimulated with soluble anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) for the indicated amount of time or left unstimulated. Phosphorylation of Zap70 and Lat was analyzed by immunoblotting in the cytosolic fraction. Densitometry analysis of the specific bands normalized to the expression of the loading control β-actin is indicated. (e) WT and Trpv1^−/− SP CD4⁺ T cells were isolated and stimulated with soluble anti-CD3 (5 μg/mL) and anti-CD28 (2 μg/mL) for 2 min or left unstimulated. Representative panels of phospho-PLCγ1 intracellular staining on gated CD4⁺TCRβ⁺ T cells are shown. (f) Cells were stimulated as in d, and phosphorylation of Erk1/2, p38 and Jnk was analyzed in the cytosolic fraction. Densitometry analysis of the specific bands normalized to the expression of the corresponding total protein is indicated. (g) NFAT-1 translocation in the nuclear fraction. (h) EMSA analysis of NF-κB translocation in the nuclear fraction. A nonspecific low molecular weight band (n.s) on the same gel was used to verify equivalent loading. Data are representative of three ore more independent experiments.

Figure 5

Genetic deletion or pharmacological inhibition of TRPV1^CD4 decreases TCR-induced cytokine production. Splenic CD4⁺ T cells were isolated from WT and Trpv1^−/− mice. (a) Cells were stimulated with anti-CD3 (10 μg/mL plate-bound) and anti-CD28 (1 μg/mL soluble) [left panel] or with PMA (25 ng/mL) and ionomycin (500 nM) [right panel] for 24 (IFN-γ, IL-17A, IL-2, TNF) or 48h (IL-10, IL-4) and cytokine production was assessed by ELISA (n = 3-6 mice/group). (b) WT and Trpv1^−/− CD4⁺ T cells were stimulated with anti-CD3+28 for 24h in RPMI supplemented with indicated concentrations of CaCl₂. Supernatants were collected and IL-2 production was measured (n = 3 mice/group). (c) OVA-loaded splenic CD11c⁺ DCs from WT mice were incubated with OVA-specific CD4⁺ T cells isolated from the spleen of WT OT-2 or Trpv1^−/− OT-2 mice. After five days of co-culture, CD4⁺ T cells were recovered, counted and equal numbers of cells were re-stimulated with anti-CD3+28 for 24h. Supernatants were collected and IFN-γ, IL-17A and IL-2 levels were measured (n = 4 mice/group). (d) WT splenic CD4⁺ T cells were pre-incubated with the indicated concentrations of BCTC (a TRPV1 inhibitor) or its vehicle (0.1% DMSO) for 30 min and stimulated with anti-CD3+28 for 24h. Supernatants were collected and IFN-γ (left-panel) and IL-2 (right-panel) production was assessed by ELISA (n = 3 mice/group). (e) Cells from the ELISA shown in d were recovered, stained for CD4, Annexin-V and 7-AAD and CD4⁺ T cell apoptosis was analyzed by flow cytometry. (f) Splenic CD4⁺ T cells from OT-2 mice were pre-treated with BCTC (1 μM) or with vehicle (0.1% DMSO) for 30 min, washed and cultured with OVA-loaded splenic DC from WT mice. After five days of co-culture, CD4⁺ T cells were processed as described in c and cytokine production was assessed (n = 4 mice/group). Data are representative of five (a, left panel), three (b-d, f) or two (a, right panel; e) independent experiments.. n.s: not significant; *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001 (two-tailed Student t-test [a], one-way ANOVA with post hoc Bonferroni [c,f] or Dunnett's [b,d,e] tests).

Figure 6

Genetic and pharmacological inhibition of TRPV1^CD4 decrease human primary CD4⁺ T cell activation. PBMCs were isolated from healthy human donors and stimulated with anti-CD3+28 (both 1 μg/mL soluble) in the presence of indicated concentrations of a TRPV1 antagonist (SB366791) or its vehicle (0.1% DMSO) for 48h. (a, b) Cells were stained for CD4, CD25 and HLA-DR, and analyzed by flow cytometry. The % of CD4⁺CD25⁺ cells and CD4⁺HLA-DR⁺ cells are indicated (right panels). (c) The supernatants were collected and IL-2 production was measured. (d) Cells were stained for CD4 and Annexin-V and CD4⁺ T cell apoptosis was analyzed by flow cytometry. (e) Freshly isolated PBMCs were nucleofected with a TRPV1 siRNA or a nontargeting (control) siRNA and stimulated with anti-CD3+28 6h after transfection. TRPV1 knockdown efficiency and (f) expression of surface activation markers, i.e., CD25 and HLA-DR were quantified on gated CD4⁺ T cells, 48h after transfection and 42h after anti-CD3+28 Abs stimulation, respectively. Transfection with TRPV1 siRNA reduced TRPV1 expression on gated CD4⁺ T cells by 40%, as well as CD25 and HLA-DR upregulation by 25% and 30%, respectively. (g) TRPV1 knockdown efficiency in a human CD4⁺ T cell clone was determined by immunoblot and IL-2 production was measured by ELISA (h) 48h after transfection and 42h after anti-CD3+28 stimulation respectively. TRPV1 expression and IL-2 production were reduced by 50% in TRPV1 knockdown CD4⁺ T cells compared to control siRNA-transfected cells. Representative panels of 4 (a-d) or 3 (e-h) independent experiments and mean ± SEM of 2 or 3 individual healthy donors are shown. n.s: not significant; *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001 (one-way ANOVA with post hoc Dunnett's test).

Figure 7

TRPV1^CD4 regulates T cell-mediated colitis. (a) Wasting disease in Il10^−/− and Il10^−/−Trpv1^−/− mice after colitis induction and synchronization by Piroxicam (PXC). Statistical analysis compared Il10^−/− and Il10^−/−Trpv1^−/− groups. (b) Representative pictures (1× and 20× objectives) of colon sections stained with H&E. Scale bar = 1 mm (overview insets), 100 μm (high-power fields). (c) Colitis score of the 3 different groups. (d) Percentage of initial body weight of Rag1^−/− recipient mice 4 weeks post-adoptive transfer with 3×10⁵ WT or Trpv1^−/− flow-sorted naive (CD4⁺CD45RB^hiCD25⁻) T cells, or with 3×10⁵ WT naive CD4⁺ T cells + 1.5×10⁵ WT T_reg cells (CD4⁺CD45RB^loCD25⁺). Statistical analysis compared WT naive CD4⁺ T cell and Trpv1^−/− naive CD4⁺ T cell groups. (e) Disease Activity Index (DAI) in the different groups. (f) Representative pictures (1× and 20× objectives) of colon sections stained with H&E. Scale bar = 1 mm (overview insets), 100 μm (high-power fields). (g) Colitis score of the different groups. (h) Cytokine concentrations in colonic explants after 24h culture. (i) Cytokine production by splenic CD4⁺ T cells isolated from Rag1^−/− recipients, 24h after re-stimulation with anti-CD3 (10 μg/mL plate-bound) and anti-CD28 (1 μg/mL soluble). One representative experiment out of two (Il10^−/− model) or three (Rag1^−/− model) is shown. Mean ± SEM (n = 6-7 [a-c], 6-8 [d-g] or 4 [h,i] mice/group). n.s: not significant; *P <0.05; **P <0.01; ***P <0.001; ****P <0.0001 (one-way [c,e,g-i] or two-way [a, d] ANOVA with post hoc Bonferroni test).

Figure 8

TRPV1 expression in non-CD4⁺ T cells does not affect colitis severity in an adoptive transfer model. (a) Wasting disease 4 weeks post-transfer. Percentage of initial body weight of Rag1^−/− or Rag1^−/−Trpv1^−/− recipient mice transferred with 3×10⁵ WT or Trpv1^−/− FACS-sorted naive (CD4⁺CD45RB^highCD25⁻) T cells. (b) Colon weight/length ratio. (c) Representative pictures (1× and 20× objectives) of colon sections stained with H&E. Scale bar = 1mm (overview insets), 100 μm (high-power fields). (d) Colitis score. (e) Cytokine production by pooled SP and MLN CD4⁺ T cells, 24h after re-stimulation with anti-CD3 (10 μg/mL plate-bound) and anti-CD28 (1 μg/mL soluble) (ELISA). Data are representative of two independent experiments. Results are expressed as mean ± SEM (n = 4 mice/group). n.s: not significant; *P <0.05 ; ***P <0.001 (one-way ANOVA with post hoc Bonferroni test).