Dual Activation of TRIF and MyD88 Adaptor Proteins by Angiotensin II Evokes Opposing Effects on Pressure, Cardiac Hypertrophy, and Inflammatory Gene Expression

Abstract

Hypertension is recognized as an immune disorder whereby immune cells play a defining role in the genesis and progression of the disease. The innate immune system and its component toll-like receptors are key determinants of the immunologic outcome through their proinflammatory response. Toll-like receptor-activated signaling pathways use several adaptor proteins of which adaptor proteins myeloid differentiation protein 88 (MyD88) and toll-interleukin receptor domain-containing adaptor protein-inducing interferon-β (TRIF) define 2 major inflammatory pathways. In this study, we compared the contributions of MyD88 and TRIF adaptor proteins to angiotensin II (Ang II)-induced hypertension and cardiac hypertrophy in mice. Deletion of MyD88 did not prevent cardiac hypertrophy and the pressor response to Ang II tended to increase. Moreover, the increase in inflammatory gene expression (Tnfa, Nox4, and Agtr1a) was significantly greater in the heart and kidney of MyD88-deficient mice when compared with wild-type mice. Thus, pathways involving MyD88 may actually restrain the inflammatory responses. However, in mice with nonfunctional TRIF (Trif(mut) mice), Ang II-induced hypertension and cardiac hypertrophy were abrogated, and proinflammatory gene expression in heart and kidneys was unchanged or decreased. Our results indicate that Ang II induces activation of a proinflammatory innate immune response, causing hypertension and cardiac hypertrophy. These effects require functional adaptor protein TRIF-mediated pathways. However, the common MyD88-dependent signaling pathway, which is also activated simultaneously by Ang II, paradoxically exerts a negative regulatory influence on these responses.

Keywords: MyD88 protein; TICAM-1 protein; angiotensin II; gene expression; hypertension; toll-like receptors.

Figure 1

Systolic blood pressure (SBP) measured during infusion of saline (black lines) or Ang II (3000 ng/kg/min, red lines) for 3 weeks in (A) WT, (B) MyD88^−/− and (C) Trif^mut mice. Values represent means ± SEM (n ≥ 3 mice in each group). The pressor response was significantly greater in the last week of Ang II infusion in MyD88^−/− than in WT (*p <0.05). Conversely, the pressor response during Ang II infusion was abrogated in the Trif^mut mice; the SBP was unchanged during infusion of saline and Ang II for the last two weeks (n.s.= p > 0.05).

Figure 2

Measurement of heart weight to body weight ratios (HW/BW ratio) after 3 weeks of saline or Ang II infusions were determined in (A) WT, (B) MyD88^−/−, and (C)Trif^mut hearts. Low (730 or 1000 ng/kg/min) or high (3000 ng/kg/min) doses of Ang II were used (ANOVA with post-hoc Tukey’s test; n ≥3; n.s. = not significant, asterisks *= P< 0.05, ***= P< 0.001). Increase in heart weight to body weight ratios with Ang II vs. saline were greater in MyD88^−/− and lesser in Trif^mut compared to WT. Linear regression analyses and slopes of dose response curves are in supplemental Table S2.

Figure 3

Comparison of pro-inflammatory gene expression in hearts from WT, MyD88^−/− and Trif^mut mice after chronic infusion (3 weeks) of saline or low dose Ang II. Fold change in RNA expression was measured by ΔΔC_T method using Gapdh RNA as a reference. Values in graphs are normalized to measurements in saline-infused mice. Tnfa= tumor necrosis factor-alpha, Nox4= NADPH oxidase 4, and Agtr1a= angiotensin receptor type 1a. (n≥3 samples each group; Asterisks show P≤0.05, n.s. = not significant). In general Ang II induced increase in gene expressions were enhanced in MyD88^−/− and reduced in Trif^mut compared to WT.

Figure 4

Comparison of pro-inflammatory gene expression in kidneys of WT, MyD88^−/− and Trif^mut after chronic infusion (3 weeks) of saline or low dose Ang II. Fold change in RNA expression was measured by ΔΔC_T method using Gapdh RNA as a reference. Values in graphs were normalized to measurements in saline-infused mice (n ≥ 3 each group; Asterisks show P ≤ 0.05, n.s. = not significant). Ang II induced increase in gene expressions were enhanced in MyD88^−/− and reduced in Trif^mut compared to WT.

Figure 5

Baseline RNA expression of TLR3, TLR4 and their adaptor proteins MyD88 and TRIF in the hearts of WT vs. MyD88^−/− and WT vs. Trif^mut mice. Comparisons of RNA expressions between WT and MyD88^−/− or WT and Trif^mut hearts were done by QPCR and ΔΔC_T method. Statistical significance was tested by unpaired t-test. Asterisks show significant differences from WT (P < 0.05) whereas n.s. denotes no significant change. In MyD88^−/− the expressions of Trif, Tlr3, and Tlr4 were increased. In Trif^mut, the expression of MyD88 was increased, Tlr4 was decreased and Tlr3 unchanged.

Figure 6

Schematic portrays Ang II induced hypertension, cardiac hypertrophy and pro-inflammatory gene expression in innate immune cells of WT, MyD88^−/− and Trif^mut mice. The dual activation of MyD88 and TRIF pathways through TLR and AT1 receptors is shown in WT cells to induce NF-κB and interferon (IFN), respectively. The TRIF-dependent and negative regulatory influence of MyD88 are highlighted.