Toll-like Receptor 9 (TLR9) Plays a Key Role in the Autonomic Cardiac and Baroreflex 1 Control of Arterial Pressure 2

25 The crosstalk between the immune and the autonomic nervous system may impact 26 the cardiovascular function. Toll-like receptors are components of the innate immune system 27 and play developmental and physiological roles. Toll-like receptor 9 (TLR9) is involved in the 28 pathogenesis of cardiovascular diseases such as hypertension and heart failure. Since such 29 diseases are commonly accompanied by autonomic imbalance and lower baroreflex 30 sensitivity, we hypothesized that TLR9 modulates cardiac autonomic and baroreflex control 31 of arterial pressure (AP). TLR9 knockout (TLR9 KO) and wild type (WT) mice were implanted 32 with catheters into carotid artery and jugular vein and allowed to recover for 3 days. After 33 basal recording of AP, mice received methyl-atropine or propranolol. AP and pulse interval 34 (PI) variability were evaluated in the time and frequency domain (spectral analysis) as well as 35 by multiscale entropy (MSE). Spontaneous baroreflex was studied by sequence technique. 36 Behavioral and cardiovascular responses to fear conditioning stress were also evaluated. AP 37 was similar between groups but TLR9 KO mice exhibited lower basal heart rate (HR). AP 38 variability was not different but PI variability was increased in TLR9 KO mice. The total 39 entropy was higher in TLR9 KO mice. Moreover, baroreflex function was found higher in 40 TLR9 KO mice. Atropine-induced tachycardia was increased in TLR9 KO mice whereas the 41 propranolol-induced bradycardia was similar to WT mice. TLR9 KO mice exhibit increased 42 behavioral and decreased tachycardia responses to fear conditioning stress. In conclusion, 43 our findings suggest that TLR9 may negatively modulate cardiac vagal tone and baroreflex in 44 mice. 45 The linear analysis of the HR dynamics, such as spectral analysis, can provide us non-invasive indices of cardiac autonomic modulation in the presence of stationary series of cardiac intervals (6). However, in situations of fast and non-repetitive variations, such as periods preceding cardiac events, these indices are less reliable because 1) spectral 327 analysis does not take into account non-linear dynamics (80, 81); 2) this analysis is based 328 only on the definition of frequency bands (55) and 3) the indexes are useful only in conditions 329 of reciprocal changes in sympathetic and parasympathetic modulations (55). HR fluctuations 330 have complex behaviors originated from non-linear dynamics and exhibit nonstationary 331 properties (28, 79). Based on physiological complexity theory (29), the healthy systems show 332 fractal variability at multiple scales and exhibit HR series with long range correlations. We analyzed the HR dynamics using the MSE method. The study of AP and/or HR 334 dynamics by MSE has been shown to predict cardiovascular outcomes and it is also 335 suggested as predictor of morbidity and mortality for several cardiovascular diseases such as 336 heart failure (94), diabetes (39), atherosclerosis (105) and others. The profile of the MSE 337 curves for HR variability for the TLR9 KO mice was different from those for the WT mice. TLR9 KO mice demonstrated an initial decrease in entropy, followed by a quick stabilization, 339 while WT mice demonstrated a steeper decrease in the entropy values, which was 340 maintained low over the time scale. Those profiles showed that irregularity, as measured by 341 sample entropy, is maintained in TLR9 KO mice as the scale increases. Uncorrelated 342 dynamics (white noise) demonstrate a monotonic decay of MSE, whereas fractal (1/f) and 343 healthy dynamics tend to show constant entropy values (16). Our results suggest that the 344 MSE from TLR9 KO mice is more similar to healthy/fractal dynamics than that from WT mice. It is common to extract a single index from MSE curves to represent complexity. The 346 most common index is the sum of the entropy values over a range of time scales (17). The sum of the The study of the role of TLR9 in the autonomic control of AP non-pathological opens