Expression of Hv1 proton channels in myeloid-derived suppressor cells (MDSC) and its potential role in T cell regulation

Abstract

Myeloid-derived suppressor cells (MDSC) are a heterogeneous cell population with high immunosuppressive activity that proliferates in infections, inflammation, and tumor microenvironments. In tumors, MDSC exert immunosuppression mainly by producing reactive oxygen species (ROS), a process triggered by the NADPH oxidase 2 (NOX2) activity. NOX2 is functionally coupled with the Hv1 proton channel in certain immune cells to support sustained free-radical production. However, a functional expression of the Hv1 channel in MDSC has not yet been reported. Here, we demonstrate that mouse MDSC express functional Hv1 proton channel by immunofluorescence microscopy, flow cytometry, and Western blot, besides performing a biophysical characterization of its macroscopic currents via patch-clamp technique. Our results show that the immunosuppression by MDSC is conditional to their ability to decrease the proton concentration elevated by the NOX2 activity, rendering Hv1 a potential drug target for cancer treatment.

Keywords: Hv1 channel; NOX2 complex; ROS; immunosuppression; myeloid cells.

Fig. 1.

H_v1 channel is expressed in mouse MDSC. Ninety-six hours after seeding bone marrow cells in the presence of GM-CSF, cell cultures were biochemically and morphologically analyzed. (A) H_v1 immunodetection by flow cytometry. A representative pseudocolor dot plot obtained by flow cytometry showed 73.1% of MDSC differentiated cells, indicating the expression of Gr-1 and CD11b markers; 97% of these immunostained cells are positive for Hv1 channels. (B) H_v1 immunodetection in MDSC cultures and MP. A band around 30 kDa corresponding to the predicted molecular weight for H_v1 monomers SDS-PAGE was evidenced by Western blot clearly in MDSC cultures, and in a highly diminished proportion also present in MP cultures. (C) Morphological characterization of Gr-1⁺ cells. Morphological analysis was done corelating cell morphology with Gr-1 (in red) and H_v1 (in green) immunostaining. The Merge column shows H_v1 channel in MDSC differentiated cells expressing Gr-1 protein; the cell nucleus was marked with DAPI (in blue) for reference. The upper lane panels show four representative round and nonadherent cells, characterized as suppressor myeloid cells. The lower lane panels show morphological differences between myeloid and dendritic phenotypes illustrating that myeloid cells were rounded. Arrows are pointing to a myeloid cell.

Fig. 2.

Electrophysiological characterization of proton currents in MDSC. (A) Representative H+ currents in membrane patches from MDSC. Micrography showing how Gr-1+ morphology was used for selecting cells to perform electrophysiological measurements (Left). MDSC proton currents from selected cells were elicited with voltage pulses of 3 s in the range from −90 to +140 mV in 10-mV increments. Currents show proton depletion (Middle). Currents were obtained at ΔpH 2 by applying voltage pulses of variable duration, from a holding potential of −90 mV to +130 mV in increments of 20 mV to avoid depletion (Right). (B and C) ΔpH dependence of H_v1 channel on MDSC. (B) Representative currents were obtained at the different ΔpH conditions, pH 5.5 in the pipette solution (pHi) and various pH values in the bath solution (pHo) (5.5, 6.5, 7.5; n = 4, 3, 3, respectively), applying the optimized variable duration pulse protocol. Note that the pulse protocols applied to lower ΔpH records had a longer duration to allow for channel activation. (C) Normalized GV curves are shown at ΔpH 2 (5.6 ± 4 mV, n = 4), ΔpH 1 (71.08 ± 1.7 mV, n = 3), and ΔpH 0 (93.24 ± 1.4 mV., n = 3). Data were fitted by a Boltzmann function. (D) Proton selectivity of MDSC currents. Selectivity of voltage-gated proton currents in MDSC was estimated from reversal potential at different ΔpH. Representative current traces at different ΔpH −0.5, 0, and 0.5 (n = 5, 5, 5, respectively) were elicited using a fast ramp pulse protocol to determine the reversal potential of voltage-gated proton currents in MDSC. Er and ΔpH relationship is shown. The experimental values were fitted by a linear regression with a −50 mV per pH unit slope. The dashed line is the theoretical value predicted for protons by the Nernst equation (−58 mV per pH unit slope). (E) MDSC proton current inhibition. Representative current traces were elicited upon depolarization from −90 to +130 mV on a cell, before (black traces) and after inhibition (blue or purple traces). (Top) Representative traces after the inhibition induced by 4-min perfusion with 10 µM ZnCl2 (blue trace). (Bottom) Current traces corresponding to a cell, perfused for 1 min with 100 µM ClGBI (purple trace).

Fig. 3.

Flow cytometry measurement of MDSC ROS production in the presence of H_v1 proton channel inhibitors. MDSC reactive species production was stimulated using 100 nM PMA. Changes in dichlorofluorescein fluorescence intensity (DCFDA), induced by the inhibitors used, 200 µM ClGBI, 1 mM ZnCl₂, or their respective vehicle controls (PBS or DMSO), were monitored for 10 min. (A) The detection of ROS production was fitted to the best curve possible via Python (red line) to better detect changes induced by the inhibitors or their respective vehicles. (B) The bar graph illustrates mean and SEM from the integrated curves for each condition, representing the cumulative ROS production. Asterisks indicate significative differences with *P < 0.005, according to nonparametric Kruskal–Wallis test. n = 3.

Fig. 4.

Pretreatment of MDSC with H_v1 proton channel inhibitors diminish their capability to suppress mitogen-induced T cell proliferation. MDSC differentiated in vitro from bone marrow precursors of C57BL/6 mice were treated for 2, 12, and 24 h with 1 mM ZnCl₂ or with 200 μM ClGBI. Untreated cells, or cells treated with the ClGBI vehicle, DMSO, were used as a control. The ability of the MDSC to suppress T cell proliferation was assessed by stimulating CFSE-stained T cells with 2 μg/mL of ConA and culturing them for 96 h in the absence or presence of MDSC in ∼1:10 of the splenocytes. (A) T cells proliferation assay. Representative histograms from flow cytometry analysis indicating the percentage of T cell proliferation for each experiment, T cell culture untreated (No Stimulus), T cells stimulated with ConA (Only ConA), stimulated T cell cocultured with MDSC (ConA + MDSC), stimulated T cells cocultured with MDSC pretreated for 2 or 24 h with ZnCl₂ [ConA + MDSC (ZnCl₂/2h)], and ConA + MDSC (ZnCl₂/24h), respectively, and the vehicle for the ZnCl₂ PBS [ConA + MDSC (PBS)]. The ClGBI vehicle was also used to induce cell proliferation, mixing with ConA + MDSC (DMSO), with ClGBI [ConA + MDSC (ClGBI/2h], or ConA + MDSC (ClGBI/24h). (B) Quantification of proliferation assay. T cell proliferation was calculated in the presence or absence of MDSC treated with ZnCl2 (Top) or ClGBI (Bottom) for 2, 12, and 24 h for three independent experiments. Proliferation comparison was done by graphing the Mean ± SE mean for each condition. Asterisks indicate significant differences *P < 0.005, by Kruskal–Wallis test.

Fig. 5.

H_v1 channel is responsible for ROS-mediated immunosuppression mechanism. (A) Modulation of ROS production by functional coupling of NOX2 and H_v1. The proliferation and response of the immune system was stimulated by the antigen presenting cells (APC) via the MHC and the TCR. When the response needs to be decreased, the MDSC produces immunosuppression in many ways, one of the main ones being ROS production by NOX2. (B) The normal mechanism for sustained ROS production, NOX2 oxidizes NADPH to NADP+ producing intracellular H+, the electron displaced ultimately forms H2O2 on the extracellular side and the action of Hv1 compensates for the accumulation of H+. (C) When Either Zn²⁺ blocks H_v1 on the extracellular side or ClGBI by the intracellular side, the accumulation of protons and consequent lowering of pH inhibits the action of NOX2, which in turn reduces the production of ROS.