Tumor necrosis factor-α enhances microvascular tone and reduces blood flow in the cochlea via enhanced sphingosine-1-phosphate signaling

Abstract

Background and purpose: We sought to demonstrate that tumor necrosis factor (TNF)-α, via sphingosine-1-phosphate signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss.

Methods: We performed intravital fluorescence microscopy to measure blood flow and capillary diameter in anesthetized guinea pigs. To measure capillary diameter ex vivo, capillary beds from the gerbil spiral ligament were isolated from the cochlear lateral wall and maintained in an organ bath. Isolated gerbil spiral modiolar arteries, maintained and transfected in organ culture, were used to measure calcium sensitivity (calcium-tone relationship). In a clinical study, a total of 12 adult patients presenting with typical symptoms of sudden hearing loss who were not responsive or only partially responsive to prednisolone treatment were identified and selected for etanercept treatment. Etanercept (25 mg s.c.) was self-administered twice a week for 12 weeks.

Results: TNF-α induced a proconstrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing sphingosine-1-phosphate receptor 2 subtype signaling (by 1 μmol/L JTE013) attenuated the effects of TNF-α in all models. TNF-α activated sphingosine kinase 1 (Sk1) and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1(G82D)) eliminated both baseline spiral modiolar artery calcium sensitivity and TNF-α effects, whereas a nonphosphorylatable Sk1 mutant (Sk1(S225A)) blocked the effects of TNF-α only. A small group of etanercept-treated, hearing loss patients recovered according to a 1-phase exponential decay (half-life=1.56 ± 0.20 weeks), which matched the kinetics predicted for a vascular origin.

Conclusions: TNF-α indeed reduces cochlear blood flow via activation of vascular sphingosine-1-phosphate signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis, and treatment.

Conflict of interest statement

Conflicts of Interest: None declared.

Figures

Figure 1. TNFα-induces reductions in stria vascularis blood flow and capillary diameter by a S1P2 receptor-dependent mechanism

Using a cochlear window preparation, alterations in guinea pig cochlear blood flow and capillary diameter were assessed in vivo. (A) Capillaries in the convex area of the cochlear second turn were visualized using epi-illumination following intravenous injection of FITC-labeled dextran. The inspection area is outlined by a white box. Superfusion of a cochlear window with TNFα (1ng/ml, 20min) reduced both (B) blood flow (measured as red blood cell velocity) and (C) capillary diameter (n=6). Pre-treatment with the S1P2 receptor antagonist JTE013 (10µmol/L for 10min) significantly attenuated both effects of TNFα.

Figure 2. TNFα and S1P stimulate vasoconstriction of spiral ligament capillaries

Changes in the capillary diameter were measured in an ex vivo preparation of capillary beds isolated from the spiral ligament of the cochlear lateral wall. (A) Capillaries were occluded on one end, opened on the other end and the red blood cells (RBC) trapped inside were visualized by laser-scanning microscopy. The capillary lumen between the occluder and the RBC was assumed to be a cylinder of constant volume: vasoconstriction (assumed to be proportional changes in diameter and length) forces the movement of RBCs towards the open end of the capillary, providing a highly-sensitive measure of vasoconstriction magnitude. (B) TNFα (1ng/ml) stimulated capillary vasoconstriction (open circles; n=7), which could be completely blocked by JTE013 pre-treatment (1µmol/L; closed circles; n=4). (C) S1P (response to 100µmol/L shown) also stimulated capillary vasoconstriction (n=9).

Figure 3. TNFα increases Ca2+ sensitivity in the spiral modiolar artery

(A) TNFα (1ng/ml, 2h) increased the Ca2+ sensitivity in isolated SMAs (diamax=89±5µm, n=5 paired observations). (B) Co-incubation with etanercept (Etc; 1µg/ml), a TNFα inhibitor, prevented the TNFα-stimulated increase in SMA Ca2+ sensitivity (diamax=91±7µm, n=5 paired observations). * denotes a significant difference (p<0.05) in the dose-response relationships.

Figure 4. The S1P2 receptor subtype is critical for TNFα-mediated Ca2+ sensitivity increase in the spiral modiolar artery

(A) Treatment of SMAs with 1µmol/L JTE013 (30min) following TNFα stimulation (1ng/ml, 2h), which increased Ca2+ sensitivity, reversed the effect of TNFα (diamax=91±4µm, n=9 paired observations). (B) In the absence of TNFα, JTE013 inhibitor treatment resulted in reduced Ca2+ sensitivity (i.e., a rightward shift in the Ca2+/tone relationship), compared to control SMAs (diamax=97±9µm, n=5 paired observations). * denotes a significant difference (p<0.05) in the dose-response relationships.

Figure 5. TNFα activates sphingosine kinase 1 to enhance spiral modiolar artery Ca2+ sensitivity

(A) Under resting conditions, GFP-Sk1 was homogenously distributed throughout the cytosol. (B) TNFα (1ng/mL; 2hrs) stimulated a redistribution of GFP-Sk1 to plasma membrane. (C) Expression of a dominant inactive Sk1 mutant dramatically reduced the resting SMA Ca2+ sensitivity (i.e., a rightward shift in the Ca2+/tone relationship), compared to controls. TNFα augmented Ca2+ sensitivity in control SMAs, but not those expressing the dominant-negative Sk1 mutant Sk1G82D (diamax Control= 90±7µm, n=7; diamax Sk1G82D=92±7µm, n=7). (D) The effects of the chemical inhibition of sphingosine kinase (dimethyl-sphingosine; DMS; 3µmol/L, 30min) were similar to that of Sk1G82D expression: it reduced resting Ca2+ sensitivity and prevented the Ca2+ sensitivity increase following subsequent application of TNFα (diamax: 87±9µm, n=7; paired observations). (E) DMS also reversed the TNFα-stimulated enhancement of SMA Ca2+ sensitivity (diamax: 86±4µm, n=5; paired observations). (F) TNFα failed to increase Ca2+ sensitivity in SMAs expressing the non-phosphorylatable, but catalytically active Sk1 mutant Sk1S225A. * denotes a significant difference (p<0.05) in the dose-response relationships.

Figure 6. Kinetics of etanercept-mediated improvement of auditory function in hearing loss patients

Displayed is the recovery kinetic from 6 clinical cases of hearing loss following off-label treatment with etanercept (Enbrel®); all patients sought treatment within 3 months of developing symptoms. In 6 out of 7 ears treated (1 patient had bilateral hearing loss), etanercept improved auditory function. Of these 6 responding ears, 50% of the maximal effect achieved after 1.5 weeks. The kinetic almost perfectly fits a one-phase exponential decay function (r2=0.9916).