Upregulation, Functional Association, and Correlated Expressions of TRPV1 and TRPA1 During Telmisartan-Driven Immunosuppression of T Cells

ABSTRACT TRPV1 and TRPA1, are known to be functionally expressed in T cells, where these two channels differentially regulate effector immune responses. Telmisartan (TM), an anti-hypertension drug, has been recently repurposed to suppress various inflammatory responses. However, the possible involvement of TRP channels during TM-driven suppression of T cells responses has not been explored yet. In this study, we investigated the potential role of TRPV1 and TRPA1 during TM-driven immunosuppression of T cells in vitro. We observed a significant elevation of both TRPV1 and TRPA1 during TM-induced immunosuppression of T cells.We found that TRPA1 activation-driven suppression of T cell activation and effector cytokine responses during TM treatment is partially, yet significantly overridden by TRPV1 activation. Moreover, the expressions of TRPV1 and TRPA1 were highly correlated in various conditions of T cell. Mechanistically, it might be suggested that TRPV1 and TRPA1 are differentially involved in regulating T cell activation despite the co-elevation of both these TRP channels’ expressions in the presence of TM. T cell activation was delineated by CD69 and CD25 expressions along with the effector cytokine levels (IFN-γ and TNF) in TM-driven suppression of T cell. These findings could have broad implications for designing possible future immunotherapeutic strategies, especially in the repurposing of TM for T cell-TRP-directed immune disorders.


Introduction
TRP channels are thermosensitive, cation-permeable ion channels with polymodal activation properties that regulate various physiological processes, including immune functions (Omari et al., 2017;Samanta et al., 2018;Zhang et al., 2023).These channels are expressed on various immune cells, such as macrophages, B cells, and T cells, playing a crucial role in cell differentiation, gene transcription, and effector responses (Acharya et al., 2021;Majhi et al., 2015;Oh-Hora & Rao, 2008;Sahoo et al., 2019;Vig & Kinet, 2009).Among these TRP channels, TRPV1 and TRPA1 are shown to be functionally associated with T cell-mediated immune responses.Pharmacological inhibition or genetic knockout of TRPV1 in T cells has been reported to reduce intracellular Ca 2+ levels, T cell activation, and effector immune functions.On the other hand, genetic deletion of TRPA1 in T cells has been shown to elevate TCR-induced Ca 2+ influx, T cell activation, and result in higher proinflammatory responses (Bertin et al., 2014(Bertin et al., , 2017;;Majhi et al., 2015;Sahoo et al., 2019).
TM is an ARB that is currently used to treat patients having hypertension (Arif et al., 2009;Gore et al., 2015;Kalikar et al., 2017).Reports indicate that TM reduces T cell responses by inhibiting the NFAT signaling and diminishing TNF-α-induced NF-κB activation (Huang et al., 2016;Nakano et al., 2009).Furthermore, the repurposing of TM has been documented to regulate various inflammatory immune diseases by its antiinflammatory or immunosuppressive effects (Arab et al., 2014;De et al., 2022;Okunuki et al., 2009).
TRPA1 and TRPV1 are non-selective cation channels that can conduct Ca 2+ -influx and recruit a series of downstream signaling events.Both TRPV1 and TRPA1 are known to be endogenously expressed in T cells (Majhi et al., 2015;Sahoo et al., 2019).Notably, various endogenous and exogenous factors can act on both TRPV1 as well as TRPA1, suggesting that, in several cases, these two channels are modulated simultaneously and/or mediate similar functions.Upregulation of TRPV1 and TRPA1 expressions during T cell activation has been reported previously (Majhi et al., 2015;Sahoo et al., 2019).However, studies regarding the involvement of the TRP channels in T cells during an immunosuppressive or anti-inflammatory stateare limited.Moreover, any potential involvement of TRP channels during TM-driven downregulation of T cell responses has yet to be investigated.
Against this backdrop, we hypothesized that there might be differential functional involvement of TRPV1 and TRPA1 in TM-induced immunosuppression of T cells.Accordingly, our aim in this study was to explorethe possible functional roles of TRPV1 and TRPA1 during TM-driven experimental immunosuppression of T cells.We examined TCR-induced T cell activation (e.g., CD69 and CD25 expression) and T cell proliferation in the presence or absence of TM.Additionally, we investigated the expressions of TRPV1 and TRPA1 on resting and activated T cells in the presence or absence of TM.Furthermore, we explored the regulation of T cell activation and effector cytokine production (e.g., IFN-γ and TNF) through TRPV1 and/or TRPA1 activation or inhibition in the presence or absence of TM.The current study might hold potential implications for immunotherapeutic strategies, particularly in the repurposing of TM in T cell-TRP-associated altered immune responses, with relevance to various health disorders and pathophysiological conditions.

Animals
Male and female C57BL/6 mice aged 6-8 weeks were sourced from the NCARE animal house facility of NISER.The experimental procedures were conducted following protocols approved (Protocol no.AH-274) by the IAEC in accordance with the CPCSEA guidelines.The mice were housed in IVCs, maintaining 12-hour day-night cycles, at a temperature of approximately 22 ± 2°C with 40-60% humidity.Prior to harvesting spleens, mice were euthanized in CO 2 chambers.In each experiment, spleens from multiple animals (ranging from 2 to 4) were collected in a 50 mL centrifuge tube containing 5 mL complete RPMI-1640 cell culture medium (RPMI-1640 + heat-inactivated FBS 10% + Penicillin (100 U/mL) + Streptomycin (0.1 mg/mL) + L-glutamine (2.0 mM) + 2-ME (55 μM)).The number of spleens was determined by the experiment plans.The 50 mL tube containing the spleens was then placed on ice until further processing.Splenocytes were isolated from these spleens within 15 to 30 min after harvesting and subjected to downstream processes.

T cell purification and culture
T cells were purified from mouse splenocytes, following a previously reported method (Kumar et al., 2022;Sahoo et al., 2018).In brief, mouse spleens were disrupted with a syringe plunger in 5 mL RPMI-1640 medium on a Petri plate and passed through a 70 μM strainer.The resulting cells were collected in a 50 mL centrifuged tube.Next, the RBCs were lysed using RBC-lysis buffer.Afterward, cells were washed with 1X PBS and resuspended in complete RPMI-1640 medium.Splenocytes were counted using a hemocytometer, and T cells were purified using a T cell isolation kit (Dynabeads TM Untouched TM Mouse T Cells Kit, Invitrogen) following the manufacturer's protocol.In brief, the splenocytes were resuspended in isolation buffer (1XPBS+ FBS 2%) after hemocytometer-based counting, and incubated with biotinylated antibodies for 20 min in a 15 mL centrifuge tube.After washing with isolation buffer, the cells were incubated for an additional 15 min with streptavidin-conjugated magnetic beads.The 15 mL centrifuge tube containing cells was then placed in a magnetic stand (DynaMag TM -15 Magnet) for 2 min, allowing T cells to be collected from the bead-free solution.The purified T cells were washed with 1X PBS, resuspended in complete RPMI-1640 medium, and subjected to downstream experiments.The purity of the isolated T cells was determined by flow cytometry (BD LSRFortessa TM , BD Biosciences) and found to be ≥ 95% pure.T cells were subsequently seeded in 48-well plates according to the experimental plans.

Experimental setups and T cell activation
To investigate different cell surface expressions (CD69 and CD25) on T cells in the presence or absence of TM, purified murine T cells were pre-incubated with either DMSO or TM (35 μM) for 1 hour.Cells were then activated with TCR (1 μg/mL of plate-bound anti-CD3 and 1 μg/mL of soluble anti-CD28 antibodies) stimulation in the presence or absence of TM and incubated for 48 hours.Next, cells were harvested, surface stained, and analyzed by flow cytometry (Figure 1a-f).
To study the T cell proliferation in the presence or absence of TM, purified murine T cells were first loaded with CFSE (3.75 μM).Next, CFSE-loaded cells were incubated with either DMSO or TM for 1 hour.Cells were subsequently activated with TCR stimulation in the presence or absence of TM and further incubated for 72 hours.Cells were then harvested and analyzed by flow cytometry (Figure 1g-h).
To examine different cell surface expressions (TRPV1 and TRPA1) on T cells in the presence or absence of TM, purified murine T cells were pre-incubated with either DMSO or TM for 1 hour.Cells were subsequently activated with either TCR or ConA (5 μg/mL) stimulation in the presence or absence of TM and incubated for 48 hours.Next, cells were harvested, surface stained, and analyzed by flow cytometry (Figure 2).
To explore the status of T cell activation (cell surface expressions of CD69 and CD25 on T cells) and cytokine production (IFN-γ and TNF) by T cells in the presence of different modulators of TRPV1 [RTX (100 nM) and BCTC (1.34 μM)] and TRPA1 [AITC (12.5 μM) and HC (100 μM)] in the presence or absence of TM, purified murine T cells were preincubated either with DMSO or with TM and/or with different functional modulators of TRPV1 and TRPA1.Next, T cells were activated with TCR stimulation and incubated for 48 hours along with the modulators in the presence or absence of TM. Cell culture supernatants were then collected to quantify cytokine levels using sandwich ELISA (Figure 4), and cells were harvested, surface stained, and subjected to flow cytometry (Figure 3).Additionally, a schematic depiction has been presented in Fig S2.
For correlation analysis (Figure 5), different experimental conditions are as follows: for Figure 5a

Flow cytometry
Cell staining and flow cytometric analysis were conducted following established protocols (Kumar et al., 2022;Majhi et al., 2015;Sahoo et al., 2019).In brief overview, cells were harvested, washed with 1X PBS, and resuspended in staining buffer (1X PBS+ BSA 1%).Cells were incubated with either fluorophore-conjugated antibodies (for CD90.2,CD69, and CD25 detection) or unconjugated antibodies (in case of TRPV1 and TRPA1 detection).The incubation period was 30 min on ice, with protection from light for fluorochrome-conjugated antibodies.Following incubation, the cells were washed with 1X PBS and resuspended in staining buffer containing 1% PFA.For samples with fluorophore-unconjugated antibodies, after primary antibody incubation, cells were washed with 1X PBS, an anti-rabbit AF488-conjugated secondary antibody was added, and incubated on ice for 30 min, with protection from light.Subsequently, cells were washed with 1X PBS and resuspended in staining buffer containing PFA (1%).c) denote the log scale from 10° to 10 5 .For the flow cytometric histograms in (g), the X-axis represents the log scale from 10 2 to 10 5 and the Y-axis denotes the count from 0 to 100.
In the case of cell proliferation assays, T cells were first stained with CFSE, washed with 1X PBS, and seeded in 48 well plates according to the experimental plans.Cells were harvested 72 hours after TCR stimulation.All flow cytometry samples were acquired using BD LSRFortessa TM flow cytometer.In each case 10,000 cells/sample were recorded, and further analyses were done using FlowJo software (BD Biosciences).FlowJo analysis was performed by gating the total cell population, excluding the low FSC-A and SSC-A signals, and then excluding the doublets.The CD90.2 positive population (T cells) against SSC-A were then selected and further cellular expressions were analyzed within the CD90.2 positive T cell population.

Elisa
Sandwich ELISAs were performed to quantify IFN-γ and TNF levels in cell culture supernatants using BD OptEIA TM mouse ELISA sets following the manufacturer's protocol (Kumar et al., 2022;Majhi et al., 2015;Sahoo et al., 2019).Cytokine concentrations (pg/mL) in culture supernatants were determined from the corresponding standard curves derived from recombinant mouse IFN-γ and TNF.

Statistical analysis
Statistical analyses were performed using GraphPad Prism 9.0 software.Data were represented as mean ± SD of 3 independent experiments(3 biological replicates).One-way ANOVA was performed for comparison between groups [Figure 1 (cell surface expressions of CD69 and CD25 on T cells in the presence or absence of TM; T cell proliferation assay in the presence or absence of TM), Figure 2 (cell surface expressions of TRPV1 and TRPA1 on T cells in the presence or absence of TM), Figure 3 (cell surface expressions of CD69 and CD25 on T cells in TRP-modulated conditions in the presence or absence of TM), Figure 4 (IFN-γ and TNF production by T cells in TRP-modulated (activated or inhibited) conditions in the presence or absence of TM)].Linear fit and correlation between points were used for the correlation analysis (Figure 5).p ≤ 0.05 was considered statistically significant.

Telmisartan (TM) suppresses TCR-induced T cell activation and proliferation
To determine the impact of TM on CD69 and CD25 levels, T cells were pre-incubated with either DMSO or 25 and 35 μM of TM, and experiments were conducted as described in the materials and methods (Figure 1a-f).No cytotoxicity was observed in the presence of 25 and 35 μM of TM; therefore, these concentrations were selected for further experiments (Fig S1A).Flow cytometric dot-plots in Figure 1a and c represent frequencies of CD69 +ve and CD25 +ve T cells along with the corresponding bar diagrams in Figure 1b and c, respectively.Figure 1e and f represent the MFI values of CD69 and CD25 expressions on T cells, respectively.Flow cytometric histograms in Figure 1g represent T cell proliferation along with the corresponding bar diagram in Figure 1h.Notably, 35 μM of TM significantly downregulated T cell activation (even in the presence of TCR, i.e., in TM+TCR condition) as the frequencies of both CD69 +ve (53.7 ± 3.90; p ≤ 0.0001) and CD25 +ve (40.3 ± 3.94; p ≤ 0.0001) T cells were reduced compared to only TCR (CD69: 72.26 ± 2.36; CD25:72.33 ± 0.93) (Figure 1a-d).This downregulation was consistent with the MFI values of CD69 (147.33 ± 7.40; p ≤ 0.001) and CD25 (50.6 ± 3.88; p ≤ 0.0001) in TM+TCR condition compared to TCR alone (CD69: 226.33 ± 18.87; CD25: 383.33 ± 11.02) (Figure 1e-f).Next, a T cell proliferation assay was carried out in the presence or absence of TM, revealing a significant downregulation of T cell proliferation in the TM+TCR condition (5.55 ± 0.99; p ≤ 0.0001) compared to only TCR (75.4 ± 1.74) (Figure 1g and h).
Moreover, the activation of both TRPV1 (by RTX) and TRPA1 (by AITC) in T cells treated with TM and TCR,i.e.,in RTX+AITC+TM+TCR (CD69: 36.6 ± 2.62,p ≤ 0.01;CD25: 20.73 ± 1.61,p ≤ 0.001) condition partially yet significantly upregulated the frequencies of both CD69 +ve and CD25 +ve T cells as compared to AITC+TM+TCR (CD69: 25.86 ± 2.25; CD25: 12.26 ± 0.65) (Figure 3a,b,f,g).However, in the absence of TM, the AITC-driven T cell suppression in the presence of TCR (i.e., in AITC+TCR) as compared to TCR alone or RTX+TCR was not reversed by the RTX+AITC+TCR condition.The partial restoration of T cell activation was only observed in T cells treated with TM and TCR, where TRPV1 activation was found to override TRPA1 activation-driven T cell suppression (i.e., RTX+AITC+TM+TCR was found to restore T cell activation as compared to AITC+TM+TCR).However, no such reversal of T cell activation was observed in TCR-treated T cells (in the absence of TM), where TRPV1 activation was not found to override the TRPA1 activation-driven suppression (i.e., RTX+AITC+TCR did not restore T cell activation as compared to AITC+TCR) of T cells.

Expression of TRPV1 correlates well with TRPA1 in T cells in different immunological conditions
Correlation analyses were performed to assess the association between surface expressions of TRPV1 and TRPA1, as well as CD69 and CD25 in various experimental conditions (as shown in Figure 5a).Frequencies of TRPA1 +ve and TRPV1 +ve T cells in six different immunological conditions remain highly correlated (r = 0.9771; p ≤ 0.001) (Figure 5a).Comparable results were observed for MFIs of TRPA1 and TRPV1 (r = 0.9903; p ≤ 0.001) (Figure 5b).
To explore the relationship between TRPV1 and TRPA1 in the context of T cell function, a standard pair of immune activation markers, i.e., a correlation between CD69 and CD25, was analyzed in sixteen different experimental conditions (as shown in Figure 5c).A high correlation between frequencies of CD69 +ve and CD25 +ve T cells (r = 0.9442; p ≤ 0.0001) and MFIs of CD69 and CD25 (r = 0.9303; p ≤ 0.0001) in various immunomodulated conditions was observed (Figure 5c and d).

Discussion
Many TRP channels, including TRPV1 and TRPA1 channels, are expressed in neurons and play a crucial role in transmitting a variety of sensory signals, such as inflammation, pain, and chemical and thermal nociception (Mickle et al., 2016;Stucky et al., 2009).TRPV1 and TRPA1 are members of the TRP family and have been reported to be functionally associated with diverse immune cells, including macrophages, dendritic cells, T cells, and NK cells (Majhi et al., 2015;Naert et al., 2021;Omari et al., 2017;Sahoo et al., 2019).The functional role of TRPV1 and TRPA1 in TCR-or ConAmediated T cell activation has been reported previously (Majhi et al., 2015;Sahoo et al., 2019).TM, an anti-hypertension drug, has been experimentally repurposed towards regulating various inflammatory disorders (De et al., 2022;Harrison et al., 2011;Okunuki et al., 2009).However, the possible involvement and association of TRPV1 and/or TRPA1 channels in TM-driven immunosuppression of T cells, if any, have not been reported until now.This study presents the initial evidence that both TRPV1 and TRPA1 participate inTM-driven suppression of T cell responses.Accordingly,our aim was to explore the possible functional involvement of TRPV1 and TRPA1 during TM-driven experimental immunosuppression of T cells.We observe that the surface expressions of TRPV1 and TRPA1 were elevated in TM-mediated immunosuppression of T cells.Moreover, TRPA1 activation-driven suppression of T cell responses during TM treatment was found to be partially yet significantly overridden by TRPV1 activation.Collectively, the data suggest that during immune activation from the basal level as well as in immunosuppressed conditions, expressions of TRPV1 and TRPA1 are upregulated, and these channels are functionally associated with T cell effector responses.
Immunosuppression promotes anti-inflammatory responses, demonstrating a critical impact in both clinical and experimental cases of altered immunity (Meneghini et al., 2021;Parlakpinar & Gunata, 2021).In this study, we used TM, an anti-hypertension drug reported to induce immunosuppression by regulating inflammatory responses (Arab et al., 2014;De et al., 2022;Nakano et al., 2009;Okunuki et al., 2009).Pre-incubation of T cells with TM effectively reduces T cell responses (CD69 and CD25 expressions, proliferation, and cytokine profile), indicating that TM-induced immunosuppression predominates over TCR-mediated T cell activation.Additionally, under TM-induced immunosuppressed conditions, activation of TRPA1 by AITC further induced immunosuppression.However, such stringent levels of immunosuppression could be partially overcome through the activation of TRPV1.Furthermore, in TM-driven immunosuppressed conditions, activation of TRPV1 by RTX was found to enhance T cell activation and effector cytokine production by overriding TRPA1-driven suppression of T cell responses.In contrast, the inhibition of TRPV1 by BCTC and TRPA1 by HC was found to induce higher immunosuppression.Therefore, it appears that the activation of endogenous TRPV1 may enhance T cell activation, especially under stringent immunosuppressive conditions.
Elevation of TRPV1 and TRPA1 during T cell activation and their functional association with T cell effector responses have been previously reported (Majhi et al., 2015;Sahoo et al., 2019).In our recent work, we demonstrated an increase in cell surface expression of TRPV1 levels in immunosuppressed T cells (Kumar et al., 2022).Additionally, our previous findings indicated an upregulationof different TRP channels during T cell activation (Acharya et al., 2021;Majhi et al., 2015;Sahoo et al., 2019) aligning well with the results presented in this study.
TRPV1 activation facilitates TCR-driven Ca 2+ -influx, T cell signaling, and effector responses in CD4 + T cells (Bertin et al., 2014).Additionally, pharmacological inhibition of TRPV1 or genetic knockout mice showed reduced disease scores, colitogenic T cell responses, and intestinal inflammation in the T cell-mediated colitis model (Bertin et al., 2014).Conversely, IL-10 −/− TRPA1 −/− mice in colitis and inflammatory bowel disease (IBD) models have been reported to develop more severe CD4 + T cell-mediated chronic inflammation as compared to mice with only IL-10 −/−10 .Moreover, TRPA1 knockout mutation has also been associated with sustained TCR-driven Ca 2+ influx, leading to Th1 (IFN-γ, IL-2 producing type 1 CD4 + helper T cells) polarization and the production of IFN-γ and IL-2 (Bertin et al., 2017).Furthermore, TRPA1 has been shown to play a suppressive role in regulating macrophage activation and proinflammatory responses (Radhakrishnan et al., 2023).In addition, immunosuppression has been reported elsewhere to upregulate intracellular Ca 2+ concentrations (Bultynck et al., 2000;Kumar et al., 2022;Racioppi et al., 2019;Shin et al., 2002) and both TRPV1 and TRPA1 have been found to play an important functional role in regulating intracellular Ca 2+ levels associated with immunosuppression (Kumar et al., 2022;Radhakrishnan et al., 2023).However, the underlying mechanisms involving immunosuppression-driven elevation of TRPV1 and TRPA1 and their possible association in regulating T cell functions require further exploration in future studies.It could be speculated that TRPV1 and TRPA1 might differentially contribute to the regulation of immune function, in addition to their upregulated expressions during cell-mediated immunosuppression.
Our data further indicate the existence of a strong correlation between surface expressions of TRPV1 and TRPA1 in T cells in different immunological conditions, such as resting, activated, and immunosuppressed states.Notably, the correlation between TRPV1 and TRPA1 surface expressions is at least the equal range, if not stronger than the CD69 and CD25, suggesting that TRPV1 and TRPA1 could be considered as contextual immunoregulatory markers.Nevertheless, the high correlation between the surface expressions of TRPV1 and TRPA1 in T cells is striking.This is also relevant as in the diverse cellular systems, both TRPV1 and TRPA1 engage in reciprocal signaling functions.
Based on these findings, we suggest that functional modulation of TRPV1 and TRPA1 could be helpful in regulating the immunosuppression of T cells.A proposed working model of the current study has been summarized in Figure 6.This study might have broad implications for the role of TRPV1 and TRPA1 in immunosuppressive diseases and might be important in designing strategies for possible future immunotherapy, especially the repurposing of TM in regulating TRP-directed cell-mediated immune responses associated with immune disorders.The current study may open future translational implications for other TRP channels in altered cell-mediated immune responses associated with various diseases.

Figure 1 .
Figure 1.Telmisartan (TM) suppresses TCR-induced T cell activation and proliferation.Flow cytometric dotplots of T cells showing frequencies of (a) CD69 and (c) CD25 positive T cells in different conditions.Bar diagrams (representing the mean ± SD of three independent experiments) showing (b) frequency of CD69 positive T cells, (d) frequency of CD25 positive T cells, (e) MFI of CD69 and (f) MFI of CD25.TM at 35 μM concentration downregulates both CD69 and CD25 levels significantly, and this concentration was used in further experiments.Flow cytometric histograms in (g) showing T cell proliferation [proliferation assay of T cells stained with CFSE] in the presence or absence of TM with or without activation along with the corresponding bar diagram in (h).Statistical analysis: one-way ANOVA; p ≤ 0.05 was considered statistically significant.ns = non-significant; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.The X and Y axes of the flow cytometric dot plots in (a) and (c) denote the log scale from 10° to 10 5 .For the flow cytometric histograms in (g), the X-axis represents the log scale from 10 2 to 10 5 and the Y-axis denotes the count from 0 to 100.

Figure 2 .
Figure 2. Elevation of cell surface TRPV1 and TRPA1 on T cells during TM-mediated immunosuppression.Flow cytometric histograms of T cells show the MFIs of (a) TRPV1 and (c)TRPA1.Bar diagrams (representing the mean ± SD of 3 independent experiments) in (b) and (d) represent the MFIs of TRPV1 and TRPA1, respectively.Percentages of TRPV1 positive and TRPA1 positive T cells are shown in (e) and (g), respectively, along with the corresponding bar diagrams in (f) and (h).Statistical analysis: one-way ANOVA; p ≤ 0.05 was considered statistically significant.ns = non-significant; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.The X and Y axes of the flow cytometric plots in (e) and (g) denote the log scale from 10° to 10 5 .

Figure 3 .
Figure 3. TRPV1 activation during TM-mediated immunoregulation overrides TRPA1-driven suppression of T cell activation.Flow cytometric dot-plots of T cells showing frequencies of (a) CD69 and (f) CD25 positive T cells in different experimental conditions.Bar diagrams (representing the mean ± SD of 3 independent experiments) showing frequency (b) and MFI (d) of CD69 positive T cells in TRPV1-and/or TRPA1-activated conditions.Bar diagrams showing frequency (g) and MFI (i) of CD25 positive T cells in TRPV1-and/or TRPA1activated conditions.Bar diagrams showing frequency (c) and MFI (e) of CD69 positive T cells in TRPV1-and/or TRPA1-inhibited conditions.Bar diagrams showing frequency (h) and MFI (j) of CD25 positive T cells in TRPV1 and/or TRPA1-inhibited conditions.In each case, one-way ANOVA was performed.p ≤ 0.05 was considered statistically significant.ns = non-significant; * = p ≤ 0.05; ** = p ≤ 0.01; *** = p ≤ 0.001; **** = p ≤ 0.0001.The X and Y axes of the flow cytometric dot plots in (a) and (f) denote the log scale from 10° to 10 5 (Oh-Hora & Rao, 2008).

Figure 5 .
Figure 5. Expression of TRPV1 correlates well with TRPA1 in T cells in different immunological conditions.(a) Correlation between frequencies of TRPV1 +ve with TRPA1 +ve T cells in different conditions is shown.(b) Correlation between MFIs of TRPV1 with TRPA1 in different conditions is shown.(c) Correlation between frequencies of CD69 with CD25 positive T cells in different conditions is shown.(d) Correlation between MFIs of CD69 and CD25 in different conditions is shown.Linear fit and correlation between points were used for the correlation analysis.In each case, the r and p values are indicated.

Figure 6 .
Figure 6.The proposed model depicting the possible involvement of TRPV1 and TRPA1 during TM-induced immunosuppression of T cells.Resting (condition (a), i.e., T cells treated with only DMSO) murine T cells were activated by TCR (i.e., T cells pre-incubated with DMSO and then stimulated with TCR) in the presence (c) or absence (b) of TM.TCR activation upregulates T cell responses relating to T cell activation markers (CD69 and CD25 expressions) and effector cytokine (IFN-γ and TNF) production as compared to resting cells (i.e., T cells treated with only DMSO).TM treatment suppresses T cell responses by downregulating T cell activation markers (i.e., CD69 and CD25 expressions) and effector cytokine (IFN-γand TNF) productions in TM+TCR condition (depicted by condition c) (i.e., pre-incubated with TM and then stimulated with TCR) as compared to only TCR activation (depicted by condition (b).Cell surface expressions of TRPV1 and TRPA1 were upregulated in TM-induced immunosuppressed T cells (c) as compared to T cells activated with only TCR stimulation (b).TRPA1 activation by AITC reduces T cell responses by reducing T cell activation markers (CD69 and CD25 expressions) and effector cytokine (IFN-γand TNF) productions in both AITC+TCR (depicted by condition (e) (i.e., T cells pre-incubated with AITC and then activated with TCR stimulation) and AITC+TM+TCR (h) (i.e., T cells pre-incubated with TM and AITC and then stimulated with TCR) conditions.TRPV1 activation by RTX during TM-mediated immunoregulation overrides TRPA1-driven suppression of T cell responses by upregulating T cell activation markers (CD69 and CD25 expressions) and effector cytokine (IFN-γ and TNF)responses i.e., in RTX+AITC+TM+TCR condition (depicted in (i), T cells pre-incubated with RTX, AITC, and TM and then stimulated with TCR) as compared to TRPA1-activated cells treated with TM and TCR, i.e., in AITC+TM +TCR condition (depicted in (h), T cells pre-incubated with AITC and TM, and then stimulated with TCR).However, in the "absence of TM", activation of TRPV1 by RTX in TCR-activated cells fails to override TRPA1 activation (by AITC)-driven suppression of T cell responses, i.e., in RTX+AITC+TCR condition (depicted in (f), i.e., T cells pre-incubated with RTX and AITC, and then stimulated with TCR) relating to T cell activation markers (CD69 and CD25 expressions) and effector cytokine (IFN-γand TNF) productions as compared to TRPA1activated cells treated with TCR, i.e., in AITC+TCR condition (depicted in (e), T cells pre-incubated with AITC and then stimulated with TCR), suggesting a possible functional involvement of TRPV1 and TRPA1 in TMmediated immunosuppression of T cells.