Toll-like receptors and myocardial inflammation

Abstract

Toll-like receptors (TLRs) are a member of the innate immune system. TLRs detect invading pathogens through the pathogen-associated molecular patterns (PAMPs) recognition and play an essential role in the host defense. TLRs can also sense a large number of endogenous molecules with the damage-associated molecular patterns (DAMPs) that are produced under various injurious conditions. Animal studies of the last decade have demonstrated that TLR signaling contributes to the pathogenesis of the critical cardiac conditions, where myocardial inflammation plays a prominent role, such as ischemic myocardial injury, myocarditis, and septic cardiomyopathy. This paper reviews the animal data on (1) TLRs, TLR ligands, and the signal transduction system and (2) the important role of TLR signaling in these critical cardiac conditions.

Figure 1

TLR signaling pathways. Upon respective ligands binding, TLRs form homo- or heterodimers and recruit one or more adaptor proteins, namely, MyD88, MAL/TIRAP, TRIF, or TRAM, to the cytoplasmic domains of the receptors through homophilic interactions between Toll/IL-1 receptor (TIR) domains present in each receptor and each adaptor. All TLRs with exception of TLR3 use the common MyD88-dependent pathway. TIRAP acts as a bridge to recruit MyD88 to TLR2 and TLR4 signaling, whereas TRIF is used in TLR3 signaling and, in association with TRAM, in TLR4 signaling. In MyD88-dependent pathway, MyD88 associates with IRAK4, IRAK1, and/or IRAK2. IRAK4 in turn phosphorylates IRAK1 and/or IRAK2 and promotes their association with TRAF6, which serves as a platform to recruit and activate the kinase TAK1. Activated TAK1 activates the IKK complex, composed of IKKα, IKKβ, and NEMO (IKKγ), which in turn catalyzes phosphorylation and subsequent degradation of IκB. IκB degradation lets NF-κB (i.e., p50/p65) free to translocate from the cytoplasma to the nucleus, where it activates multiple gene expression. The transcription factor IRF7 is activated as the downstream signaling molecule of TLR 7, 8, and 9. It is directly phosphorylated by IRAK1 and then translocates into the nucleus to induce the expression of type I IFN and IFN-inducible genes. In the Trif-dependent pathway, Trif interacts with TRAF3 to activate TBK1 and IKKi, resulting in the dimerization and activation of IRF3, which then translocates into the nucleus activating the transcription of type I IFN and IFN-inducible genes.

Figure 2

Myocardial neutrophil recruitment after I/R in MyD88^−/− and Trif^−/− mice. Twenty-four hours after 60 min of left anterior descending coronary artery (LAD) ligation, the hearts were isolated, perfused, and digested. After removal of the large cardiomyocytes through filtration, 50% of total cells were loaded onto flow cytometry and gated on Gr-1 and CXCR2. (a) Total Gr-1+ cells as measured by flow cytometry from the hearts subjected to I/R in MyD88^−/− mice. Each error bar represents mean ± SD of 4 mice. A small number of neutrophils were recovered from the sham-operated hearts as indicated by the line. (b) A representative example of flow cytometry plots of myocardial infiltrating cells from sham, WT-I/R, and MyD88^−/−-I/R mice. (c) Total Gr-1+ cells as measured by flow cytometry from the hearts subjected to I/R in Trif^−/− mice. Each error bar represents mean ± SD of 3 mice. A small number of neutrophils were recovered from the sham-operated hearts as indicated by the line. (d) A representative example of flow cytometry plots of myocardial infiltrating cells from WT-I/R and Trif^−/−-I/R mice. FSC, forward scatter; SSC, side scatter. (Feng et al., [100], used with permission).

Figure 3

Decreased MI sizes in MyD88-knockout (KO) and KO→WT chimeric mice compared with wild-type (WT) and WT→WT chimeric mice. Mice were subjected to 30 min of ischemia and 24 h of reperfusion. At the end of reperfusion, animals were euthanized, and area-at-risk (AAR) and MI were analyzed. (a) Representative of triphenyltetra zolium chloride (TTC) staining (bottom) and fluorescent microsphere distribution (top) of myocardial sections from the 4 groups of mice. The nonischemic area is indicated by red fluorescent staining, area at risk (AAR) by area devoid of red fluorescent light, and infarct area by white. (b) Cumulative data of AAR/left ventricle (LV). (c) Cumulative data of MI/AAR. Each error bar represents mean ± SD of 6–9 mice. (Feng et al., [100], used with permission).

Figure 4

Schematic diagram of cardiac infarct size (blue region) after acute ischemia and reperfusion (I/R) and neutrophil CXCR2 downregulation by deletion of myeloid differentiation factor 88 (MyD88) globally (right top) and targeted to leukocytes only (right bottom). (Schmid-Schönbein, [103], used with permission).

Figure 5

Myocarditis in TLR3^−/− mice infected with CVB3. (a) Histopathologic results of hearts collected from TLR3^−/− and wild-type mice 12 days after CVB3 infection was evaluated with Masson's trichrome staining. Data represent eight mice per group. (b) Immunofluorescence of hearts from TLR3^−/− or wild-type mice 9 days after CVB3 infection followed by anti-CVB3 antibody staining. Data represent three mice per group. (c),(d) Real-time RT-PCR analysis of the expression of positive-strand CVB3 RNA (c) or the indicated cytokine genes (d) in hearts of TLR3^−/− and wild-type mice on the indicated days after CVB3 infection. Data are presented as mean ± SD of triplicate determinations. All experiments were performed more than twice with similar results. **P < 0.01; *P < 0.05. (Negishi et al., [108], used with permission).

Figure 6

TLR2^−/− mice have improved cardiomyocyte function after polymicrobial sepsis. Wild-type (WT) and TLR2^−/− mice underwent sham or cecum ligation and puncture (CLP) procedures. Twenty-four hours later, the hearts were harvested and cardiomyocytes were isolated. A, Representative tracing of sarcomere shortening and Ca²⁺ transients in cardiomyocytes isolated from WT (a, b) and TLR2^−/− (c, d) mice subjected to either sham (a, c) or CLP (b, d) surgeries. B, Accumulated data of sarcomere shortening and Ca²⁺ transients. Each error bar represents mean ± SE. The data in each group were recorded from 16 to 27 single adult cardiomyocytes isolated from more than four mice. *P < 0.05 versus WT sham; **P < 0.01 versus WT CLP; ^‡ P < 0.001 versus WT sham. KO, knockout (TLR2^−/−). (Zou et al., [124], used with permission).