Angiotensin II-induced hypertension and cardiac hypertrophy are differentially mediated by TLR3- and TLR4-dependent pathways

Abstract

Toll-like receptors (TLR) are key components of the innate immune system that elicit inflammatory responses through the adaptor proteins myeloid differentiation protein 88 (MyD88) and Toll-interleukin receptor domain-containing adaptor protein-inducing interferon-β (TRIF). Previously, we demonstrated that TRIF mediates the signaling of angiotensin II (ANG II)- induced hypertension and cardiac hypertrophy. Since TRIF is activated selectively by TLR3 and TLR4, our goals in this study were to determine the roles of TLR3 and TLR4 in mediating ANG II-induced hypertension and cardiac hypertrophy, and associated changes in proinflammatory gene expression in heart and kidney. In wild-type (WT) mice, ANG II infusion (1,000 ng·kg^-1·min^-1 for 3 wk) increased systolic blood pressure and caused cardiac hypertrophy. In ANG II-infused TLR4-deficient mice (Tlr4^del), hypertrophy was significantly attenuated despite a preserved or enhanced hypertensive response. In contrast, in TLR3-deficient mice (Tlr3^-/-), both ANG II-induced hypertension and hypertrophy were abrogated. In WT mice, ANG II increased the expression of several proinflammatory genes in hearts and kidneys that were attenuated in both TLR4- and TLR3-deficient mice compared with WT. We conclude that ANG II activates both TLR4-TRIF and TLR3-TRIF pathways in a nonredundant manner whereby hypertension is dependent on activation of the TLR3-TRIF pathway and cardiac hypertrophy is dependent on both TLR3-TRIF and TLR4-TRIF pathways. NEW & NOTEWORTHY Angiotensin II (ANG II)-induced hypertension is dependent on the endosomal Toll-like receptor 3 (TLR3)-Toll-interleukin receptor domain-containing adaptor protein-inducing interferon-β (TRIF) pathway of the innate immune system but not on cell membrane localized TLR4. However, ANG II-induced cardiac hypertrophy is regulated by both TLR4-TRIF and TLR3-TRIF pathways. Thus, ANG II-induced rise in systolic blood pressure is independent of TLR4-TRIF effect on cardiac hypertrophy. The TLR3-TRIF pathway may be a potential target of therapeutic intervention.

Keywords: MyD88; TLR3; TLR4; TRIF; Toll-like receptors; angiotensin II; cardiac hypertrophy; hypertension; innate immune system.

Fig. 1.

A: measurement of systolic blood pressure (SBP) during saline (shaded circles) or angiotensin II (ANG II) (closed circles, 1,000 ng·kg⁻¹·min⁻¹) infusions for 3 wk in wild-type mice (WT, n = 6, 8), mice with TLR4 deletion (Tlr4^del, n = 9, 8), and TLR3 knockout (Tlr3^−/−, n = 6, 6) mice. Values represent means ± SE. Dotted horizontal line corresponds to the peak SBP in WT and is displayed for comparison with SBP of Tlr4^del and Tlr3^−/− mice. Statistically significant increases in SBP by ANG II in WT and Tlr4^del are marked by * (P ≤ 0.05 for values compared with the saline infusion on corresponding days, using unpaired t-test). Increase in SBP in ANG II-infused Tlr3^−/− was not different from that seen with saline throughout the 3-wk infusion period. B: comparison of the effect of ANG II on SBP (ΔSBP) in WT, Tlr4^del, and Tlr3^−/− during the first 9 days (1st phase) and last 10 days (2nd phase) of infusion. These results were analyzed by one-way ANOVA and Tukey’s multiple comparison test (*statistically significant changes, P ≤ 0.05; n.s., not significant). Compared with the WT, response to ANG II infusion in Tlr4^del was significantly greater during the 1st phase but not during the 2nd phase, whereas response in Tlr3^−/− was suppressed during both 1st and 2nd phases of ANG II infusion. C: peak SBP in ANG II-infused WT, Tlr4^del, and Tlr3^−/− mice. Peak SBP values in ANG II-infused WT and Tlr4^del mice were significantly greater than saline-infused mice but were not different between saline- and ANG II-infused Tlr3^−/− (*significant difference from saline-infused values, unpaired t-test, P ≤ 0.05; n.s., not significant).

Fig. 2.

A: cardiac hypertrophy measured as heart weight/ body weight (HW/BW) ratios after 3 wk of either saline or angiotensin II (ANG II) infusion in wild-type (WT; n = 6 for saline and n = 8 for ANG II), Tlr4^del (n = 9 for saline and n = 6 for ANG II), and Tlr3^−/−mice (n = 6 in each group). HW/BW ratio was significantly greater in ANG II-infused vs. saline-infused WT mice (38% increase), but the increase in ANG II-infused vs. saline-infused Tlr4^del mice was significantly less than in WT (15% increase, P = 0.004). There was no significant difference in the HW/BW ratio between saline- and ANG II-infused Tlr3^−/− mice at the end of the infusion period with ANOVA and post hoc Tukey’s multiple comparison test for ANG II-infused HW/BW ratios. *P < 0.05. B: graph representing cross-sectional areas of left ventricular cardiomyocytes from WT (saline 197.3 ± 1.9 µm², n = 417 cells; ANG II 269.7 ± 13.5 µm², n = 537 cells), Tlr4^del (saline 186.0 ± 6.4 µm², n = 260 cells; ANG II 178.6 ± 10.4 µm², n = 287), and Tlr3^−/− (saline 202.8 ± 12.3 µm², n = 225; ANG II 207.5 ± 19.3 µm², n = 311). Cross sections obtained from three mice in each group at the end of 3-wk infusions of saline or ANG II were stained with hematoxylin-eosin. Compared with saline infusion, ANG II infusion caused significant increases in cross-sectional area of cardiac myocytes in WT hearts but not in Tlr4^del or Tlr3^−/− hearts. (*P < 0.05 for unpaired t-tests; n = 3 mice each group). C: thrombi were seen histologically in hearts from ANG II-infused WT mice (four hearts examined) but not in the Tlr4^del or Tlr3^−/− hearts (three hearts of each strain examined). Thrombi were associated with inflammation that is marked by arrows showing infiltration of leukocytes (purple nuclei) extending into adjacent cardiac walls. Hematoxylin-eosin stains, magnification ×100. n.s., not significant; RV, right ventricle.

Fig. 3.

Real-time PCR comparisons of changes in inflammation-related cardiac gene expression induced by angiotensin II (ANG II) infusion in wild-type (WT), Tlr4^del, and Tlr3^−/−mice. RNA expression was measured by ΔΔCt method using Gapdh RNA as a reference. Values displayed represent ANG II-induced changes in expression relative to values in saline-infused mice. Increases with ANG II seen in all cardiac genes in WT were lesser in Tlr4^del except for Il1b expression that was greater. All gene expressions were more uniformly attenuated in Tlr3^−/− and particularly Il1b that was actually decreased. *Significant changes (P ≤ 0.05; n.s., not significant) by unpaired t-tests (n = 3 samples in each group). Il1b, interleukin-beta 1 (P–R); Il6, interleukin-6 (M–O); Mmp9, matrix metalloprotease 9 (J–L); Nppa, atrial natriuretic peptide (A–C); Nox4, NADPH-oxidase 4 (D–F); Tnf, tumor necrosis factor-alpha (G–I).

Fig. 4.

A: real-time PCR comparison of angiotensin II (ANG II)-induced proinflammatory gene expression in kidneys of wild-type (WT), Tlr4^del, and Tlr3^−/− mice after 3-wk infusion of ANG II. Relative change in RNA expression was determined by ΔΔCt method using Gapdh RNA expression as a reference. Values of ANG II-induced RNA expression relative to their expression in kidneys from saline-infused mice were compared by t-test (n ≥ 3 samples in each group, *P < 0.05 by t-tests; n.s., not significant). Significant increases with ANG II infusion were seen in the expression of Il6 in WT and of both Il6 and Tgfb2 in Tlr4^del. In contrast, there were decreases in Nox4 and Tgfb2 expression in Tlr3^−/−. These changes would coincide with the loss of pressor response in Tlr3^−/−. B: immunoblot of NOX4 and GAPDH proteins in kidneys of saline- or ANG II-infused WT, Tlr4^del, and Tlr3^−/− mice. Equal amounts of total protein (30 µg per lane) were resolved on a bis-tris gel and immunodetected using an anti-NOX4 or anti-GAPDH antibody and enhanced chemiluminescence. Lanes 1 and 2 (WT saline), lanes 3 and 4 (WT ANG II), lanes 5 and 6 (Tlr4^del saline), lanes 7 and 8 (Tlr4^del ANG II), lanes 9 and 10 (Tlr3^−/− saline), and lanes 11 and 12 (Tlr3^−/− ANG II) show samples from individual mice. Band intensities were measured within the linear dynamic range of signals by Gel Imager (Bio-Rad). A representative image of an X-ray film exposure is shown here. NOX4 band intensities were quantified and normalized to the intensities of GAPDH band in the corresponding samples. C: graph showing comparison of NOX4 protein between the kidneys of saline- and ANG II-infused WT, Tlr4^del, and Tlr3^−/− mice. Mean values of the ratios of NOX4 to GAPDH band intensities from kidneys of saline- or ANG II-infused mice are presented. Changes are compared from saline-infused samples of the same genotype. Increase in NOX4 in samples of ANG II-infused WT mice was significantly greater than in ANG II-infused Tlr4^del or Tlr3^−/− (unpaired t-test). There was no significant difference between NOX4 protein from ANG II-infused Tlr4^del and Tlr3^−/−. *Statistically significant difference (P ≤ 0.05; n.s., not significant). Il1b, interleukin-beta 1; Il6, interleukin-6; Nox4, NADPH-oxidase 4; Tgfb2, transforming growth factor beta 2.

Fig. 5.

Expression of mRNA of Tlr3, Tlr4, and Agtr1a in organs of wild-type (WT) mice relative to the spleen. Relative expression in heart, kidney, aorta, and brain were compared with expression in spleen by ΔΔCt method using Gapdh RNA expression as reference (n = 3 in each group). RNA expression in different tissues was analyzed by one-way ANOVA and Tukey’s multiple comparisons test. *Significant differences from spleen (P ≤ 0.05). Agtr1a, angiotensin II receptor type 1a; Tlr3, Toll-like receptor 3; Tlr4, Toll-like receptor 4.

Fig. 6.

Comparison of baseline expression of Agtr1a in the hearts of wild-type (WT), Tlr4^del, and Tlr3^−/− mice. Expression of Agtr1a in the hearts of WT and Tlr4^del was similar but markedly increased in the hearts of Tlr3^−/− mice (n = 3 in each group, *P ≤ 0.05, one-way ANOVA and Tukey’s post hoc test; n.s., not significant). Agtr1a, angiotensin II receptor type 1a.

Fig. 7.

Effect of angiotensin II (ANG II) infusion on cardiac and renal expression of Tlr3 and Tlr4. ANG II infusion for 3 wk did not alter the expression of Tlr3 or Tlr4 in hearts or kidneys of Tlr4^del or Tlr3^−/− mice, respectively. Only hearts from ANG II-infused wild-type (WT) mice showed significant increase in Tlr4 expression. (n = 3 each group, *P < 0.05). n.s., not significant; sal, saline; Tlr3, Toll-like receptor 3; Tlr4, Toll-like receptor 4.

Fig. 8.

Schematic of proposed model of differential effects of angiotensin II (ANG II) on hypertension and cardiac hypertrophy. TLR3 mediated TRIF activation is required for ANG II hypertension, whereas TLR-TRIF is not. This is likely due to intrinsic differences between TLR4-TRIF and TLR3-TRIF interactions. The former requires TRAM as adaptor protein to activate TRIF, whereas TLR3 directly interacts with TRIF. Moreover, TLR4 activates an additional adaptor protein, MyD88, which in contrast to TRIF, exerts a restraint on ANG II hypertension. In contrast, ANG II-induced cardiac hypertrophy is dependent on the TRIF-mediated pathway by both TLR4 and TLR3. DAMP, damage-associated molecular patterns; MyD88, myeloid differentiation protein 88; TLR, Toll-like receptor; TRAM, TRIF-related adaptor molecule; TRIF, Toll-interleukin receptor domain-containing adaptor protein-inducing interferon-β.