ATP-mediated Events in Peritubular Cells Contribute to Sterile Testicular Inflammation

Abstract

Peritubular myoid cells, which form the walls of seminiferous tubules in the testis, are functionally unexplored. While they transport sperm and contribute to the spermatogonial stem cell niche, specifically their emerging role in the immune surveillance of the testis and in male infertility remains to be studied. Recently, cytokine production and activation of Toll-like receptors (TLRs) were uncovered in cultured peritubular cells. We now show that human peritubular cells express purinergic receptors P2RX4 and P2RX7, which are functionally linked to TLRs, with P2RX4 being the prevalent ATP-gated ion channel. Subsequent ATP treatment of cultured peritubular cells resulted in up-regulated (pro-)inflammatory cytokine expression and secretion, while characteristic peritubular proteins, that is smooth muscle cell markers and extracellular matrix molecules, decreased. These findings indicate that extracellular ATP may act as danger molecule on peritubular cells, able to promote inflammatory responses in the testicular environment.

Figure 1

Expression of purinoceptors P2RX4 and P2RX7 in peritubular cells. (a) Expression of P2RX4 and P2RX7 mRNA was revealed in HTPCs stemming from four individual patients (1–4) and in the human testis (+). Patient-derived HTPCs were additionally screened for the presence of smooth muscle cell markers ACTA2 and CNN1 and absence of the mast cell marker TPSAB1. Negative controls: non-reverse transcription control (−RT), non-template control (−). (b) Immunoblotting confirmed P2RX4 and P2RX7 expression in HTPCs from three different patients (a–c). Note that gel/blot images were cropped and full-length gels/blots are presented in Supplementary Fig. 5. (c) Basal P2RX4 (n = 8) and P2RX7 (n = 8/6) receptor mRNA levels at 6 h and 24 h varied between cells derived from individual patients, but also ACTA2 levels (n = 8) did not remain constant. Data are geometric mean with 95% confidence interval. (d) P2RX4 expression was detected in peritubular cells, germ cells and interstitial tissue, while P2RX7 expression was found in peritubular cells and vessels (not shown) solely. P2RX4 expression in the tubular wall overlapped with SMA and CNN1 expression and absence of tryptase staining in consecutive sections. Insets: Negative controls (pre-adsorption for P2RX4, omission of primary antibody for P2RX7); Bars = 20 µm.

Figure 2

Extracellular ATP stimulates HTPCs. (a) Phase-contrast micrograph depicting cultured HTPCs. A single cell is targeted by a patch pipette. (b_i) Original current traces from representative whole-cell patch-clamp recordings (V_hold = −40 mV; S₁, S₄) from cultured HTPC challenged with ATP at increasing concentrations (10, 1,000 µM) and exposure durations (1 s, left and middle; 5 s right). Note that prolonged stimulation clearly revealed current desensitization. For clarity, currents were smoothed according to a ‘box 7’ algorithm (Igor Pro software) (b_ii) Bar graph quantifying the percentage of ATP-sensitive HTPCs. (c) Quantification of recordings as shown in (b). Bar chart depicting peak current amplitude measurements (mean ± SEM) in response to variable [ATP]_ex (10, 100 and 1,000 µM). Numbers of experiments are indicated above bars. (d) Average maximal I–V curve (black trace) in response to 100 µM ATP (n = 4). Grey shadows indicate SEM. Inset shows mean currents at −80 mV and +80 mV, revealing substantial inward rectification. Representative current–voltage relationships (e) and current time course (f) in response to 100 µM ATP. Inset (e): Command voltage ramp, repeated at 2 Hz. (f) Representative plots of current measurements at −80 mV (black dots) and +80 mV (empty circles), respectively, over time. When challenged with 100 µM ATP, a fast, but relatively small inward current develops and shows a transient peak followed by apparent desensitization.

Figure 3

ATP-dependent Ca²⁺ mobilization in HTPCs. (a and b) HTPC [Ca²⁺]_c signals in response to increasing ATP concentrations (30, 300 and 1,000 µM; 1 s) were monitored by ratiometric fluorescence imaging in fura-2/AM-loaded cells. (a) Pseudocolour single frame images illustrate relative [Ca²⁺]_c at different time points (rainbow 256 colourmap; blue = low Ca²⁺/red = high Ca²⁺). (b) Original traces depict the integrated fluorescence ratio f₃₄₀/f₃₈₀ of representative cells in user-defined regions of interest (ROIs; colour as in (a)) as a function of time. Note the discontinuous ordinate (//). (c and d) Average dose-response (c) and dose-‘recruitment’ (d) curves depict peak elevations in [Ca²⁺]_c (c) and the percentage of responsive HTPCs (d) upon exposure to increasing ATP concentrations, ranging from 0.1 µM to 1,000 µM. The threshold concentration for activation is ~3 µM, half-maximal activation/recruitment (EC₅₀) is observed at 41 µM (c) and 15 µM (d), respectively, and saturated signals are induced by ATP concentrations of ≥300 µM. Sigmoid dose-response curves were calculated using the Hill equation. Individual data points in (c) show means ± SEM (n = 36–158).

Figure 4

Pharmacological signature of ATP-dependent HTPC Ca²⁺ signals. (a–d) Representative original traces (f₃₄₀/f₃₈₀ versus time) of [Ca²⁺]_c transients recorded from individual HTPCs challenged with ATP (1000 µM (a,b); 30 µM (c,d); 5 s; arrows) under control conditions and during different biophysical/pharmacological treatments. Signals are abolished in absence of external Ca²⁺ (a), essentially unaffected by the P2RX7 receptor antagonist A438079 (10 µM (b)), and strongly or partly reduced in presence of PPADS (c) or TNP-ATP (d), respectively. Treatment duration (pre-incubation) is indicated by horizontal bars. (e) Bar chart quantifying the effects of different conditions on [Ca²⁺]_c response amplitudes (black bars). Data are means ± SEM, normalized to control conditions (i.e., prior stimulation; white bars). Numbers of cells are indicated above bars. Asterisks (*) denote statistical significance, p < 0.05 (paired t-test).

Figure 5

ATP treatment affected characteristic peritubular markers and inflammation-associated genes. (a) After ATP treatment of HTPCs P2RX4 (n = 8) and P2RX7 (n = 8/6) mRNA levels were significantly down-regulated. (b) mRNA levels of smooth muscle cell markers ACTA2 (n = 8) and CNN1 (n = 8) were significantly decreased by ATP treatment. (c) Stem cell niche regulatory factors CXCL12 (n = 8) and GDNF (n = 8/6) mRNAs were marginally influenced in a time-dependent manner. (d) Inflammation-associated genes IL1B (n = 8/5), IL6 (n = 8), IL33 (n = 8), CCL2 (n = 8) and CCL7 (n = 8/6) mRNA levels were increased after ATP treatment. Data are means ± SEM after 6 h and 24 h, normalized to control conditions. Asterisks denote statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001 (one-sample t-test).

Figure 6

ATP treatment influences cytokine secretion and ECM protein expression. (a) CXCL5 and CCL7 levels were numerically, but not statistically significantly elevated after ATP treatment of HTPCs, whereas IL6 and CCL2 levels were unaltered (n = 4 each). (b) At mRNA level, CXCL5 increased statistically significantly (n = 8/6). (c) ATP treated supernatants of HTPCs exhibited a statistically significant decrease in IGFBP3, SPP1 and a numerical decrease in THBS1 (n = 4 each) protein levels. (d) Decrease of IGFBP3 (n = 8), SPP1 (n = 8/6) and THBS1 (n = 8) was recapitulated on mRNA level, yet only the later two reached statistical significance. (e) Down-regulated candidate proteins after ATP treatment clustered for enrichment in pathways or GO terms related to ECM organization and collagen metabolism. Collagens were the most highly abundant proteins within this subset. (f) ATP-induced decrease of collagens was confirmed by reduced mRNA levels of COL1A1, COL1A2, COL3A1, COL4A2, COL6A2 and LOX (n = 8 each) after 6 h, but not 24 h of treatment. Data are means ± SEM (protein: after 48 h; mRNA: after 6 h and 24 h), normalized to control conditions. Asterisks denote statistical significance, *p < 0.05, **p < 0.01, ***p < 0.001 (one-sample t-test).