The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation

Abstract

The Kv1.3 channel has been widely demonstrated to play crucial roles in the activation and proliferation of T cells, which suggests that selective blockers could serve as potential therapeutics for autoimmune diseases mediated by T cells. We previously described that the toxin mimic FS48 from salivary gland of Xenopsylla cheopis downregulates the secretion of proinflammatory factors by Raw 264.7 cells by blocking the Kv1.3 channel and the subsequent inactivation of the proinflammatory MAPK/NF-κB pathways. However, the effects of FS48 on human T cells and autoimmune diseases are unclear. Here, we described its immunomodulatory effects on human T cells derived from suppression of Kv1.3 channel. Kv1.3 currents in Jurkat T cells were recorded by whole-cell patch-clamp, and Ca²⁺ influx, cell proliferation, and TNF-α and IL-2 secretion were measured using Fluo-4, CCK-8, and ELISA assays, respectively. The in vivo immunosuppressive activity of FS48 was evaluated with a rat DTH model. We found that FS48 reduced Kv1.3 currents in Jurkat T cells in a concentration-dependent manner with an IC₅₀ value of about 1.42 μM. FS48 also significantly suppressed Kv1.3 protein expression, Ca²⁺ influx, MAPK/NF-κB/NFATc1 pathway activation, and TNF-α and IL-2 production in activated Jurkat T cells. Finally, we show that FS48 relieved the DTH response in rats. We therefore conclude that FS48 can block the Kv1.3 channel and inhibit human T cell activation, which most likely contributes to its immunomodulatory actions and highlights the great potential of this evolutionary-guided peptide as a drug template in future studies.

Keywords: FS48; Jurkat T cells; Xenopsylla cheopis; autoimmune disease; immunomodulator; potassium channel.

Figure 1

Modulation of FS48 on endogenous voltage-gated potassium channels.A, representative traces of MgTx and different concentrations of FS48 suppressing the Kv1.3 currents in Jurkat T cells. Currents were elicited by applying 200 ms depolarization pulses from a holding potential of −70 mV to +40 mV in Jurkat T cells. B, concentration–response curve of FS48 inhibiting Kv1.3 currents in Jurkat T cells. Currents were normalized to the control and fitted by a Hill equation. C, current–voltage relationships (I-V). Test potentials were ranged from −50 mV to +40 mV with 10 mV increment steps. Y-axis represents the currents at different activation potential and normalized to the bath current at +40 mV in the present (red) or absent (black) of FS48; The solid lines represent the average Boltzmann sigmoidal fits. Data are shown as mean ± SD (n ≥ 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 compared with bath group.

Figure 2

Effects of FS48 on mRNA and protein expression of Kv1.3 channel.A, the viability of Jurkat T cells incubated with indicated concentrations of FS48 for 24 h. B, the relative expression analysis of KCNA3 mRNA in the presence and absence of FS48 and MgTx by qRT-PCR. C, Kv1.3 protein expression analysis of Kv1.3 channel. The cells were treated with PMA/ionomycin (50 ng/ml; 1 μg/ml) for 24 h after incubated with 3 μM FS48 and 100 nM MgTx for 1 h and then were collected for Western blot analysis. D, the ratios of Kv1.3 proteins to GAPDH. Quantity One software (Bio-Rad) was used for band density analysis. Data are shown as mean ± SD (n ≥ 3). ^#p < 0.05, compared with negative control group. ∗p < 0.05, compared with model group.

Figure 3

Effect of FS48 on the Ca²⁺influx to Ca²⁺-depleted Jurkat T cells.A, flow cytometry analysis of the concentration of intracellular Ca²⁺ in Jurkat T cells. B, the summarized time course of the suppressed Ca²⁺ influx to Ca²⁺-depleted Jurkat T cells by FS48. Jurkat T cells were incubated with Fluo4-AM, resuspended in Ca²⁺ -free Ringer solution, and treated with 0, 0.1, 1, and 10 μM FS48 for 20 min before the intracellular Ca²⁺ release, and Ca²⁺ influx was elicited by TG and CaCl₂, respectively. The fluorescence signals were read using microplate spectrophotometer. C, the ΔF/F₀ value of the peak intracellular Ca²⁺ release response stimulated by TG. D, the ΔF/F₀ value of the maximum Ca²⁺ influx response after 2 mM CaCl₂ application. All data are presented as mean ± SD (n ≥ 3). ∗p < 0.05, ∗∗∗p < 0.001, compared with negative control group.

Figure 4

Effect of FS48 on the activation of MAPK/NF-κB/NFATc1 pathway. Jurkat T cells were pretreated with 3 μM FS48 and 100 nM MgTx for 1 h, then the cells were stimulated with PMA/ionomycin (50 ng/ml; 1 μg/ml) for 4 h before collected for Western blot analysis. A and B, representative Western blot analysis of NF-κB p65 and NFATc1 nuclear translocation, ERK, JNK, and p38 phosphorylation. C, the ratios of p65 and NFATc1 protein to Histone H3 and phosphorylated ERK, JNK, and p38 to corresponding total protein. Quantity One software (Bio-Rad) was used for band density analysis. All data are shown as mean ± SD (n ≥ 3). ^###p < 0.001, compared with negative control group; ∗∗∗p < 0.001 compared with model group.

Figure 5

Effect of FS48 on the secretion of TNF-α and IL-2 in Jurkat T cells stimulated with PMA/ionomycin.A, effect of FS48 (1, 3, 10, 30 μM) and 100 nM MgTx on the proliferation of Jurkat T cells stimulated with PMA/ionomycin. B, knockdown of Kv1.3 channel expression with different siRNA. C and D, the mRNA production of TNF-α and IL-2. E and F, the inhibition rate of TNF-α and IL-2 secretion in Jurkat T cells. G and H, the inhibition rate of TNF-α and IL-2 in Jurkat T cells after knockdown Kv1.3. All data are presented as mean ± SD (n ≥ 3). ^###p < 0.001, compared with negative control group; ∗p < 0.05, ∗∗∗p < 0.001 compared with model group.

Figure 6

DTH immunosuppressive effects of FS48.A, photographic images of rat ear edemas. Photographs at 24 h postinjection show the gross pathology of ear from saline (a and d), OVA plus saline (b and e), OVA plus 2.5 mg/kg FS48 (c and f) groups. The top and lateral views of ears are displayed in parts a–c and d–f, respectively. B, the thickness increase between the OVA-challenged ear and the saline-treated ear in each group (n = 6) 24 h after the second challenge. C and D, the summarized secretion of TNF-α and IL-2 in the ear tissue. E, histology analysis of rat auricle tissue sections in vehicle (Panel a), OVA-treated (Panel b) and OVA +2.5 mg/kg FS48-treated DTH rats (Panel c) in 24 h (Original magnification, ×100). Data were showed as means ± SD, ^###p < 0.001 compared with vehicle group; ∗p < 0.05 and ∗∗∗p < 0.001 compared with model group.