Myocardial ischaemia-reperfusion (MIR) triggers a sterile inflammatory response important for myocardial healing, but which may also contribute to adverse ventricular remodelling. Such inflammation is initiated by molecular danger signals released by damaged myocardium, which induce innate immune responses by activating toll-like receptors (TLRs). Detrimental roles have been recently reported for TLR2, TLR3 and TLR4. The role of other TLRs is unknown. We therefore evaluated the role of TLR5, expressed at high level in the heart, in the development of myocardial damage and inflammation acutely triggered by MIR. TLR5−/− and wild-type (WT) mice were exposed to MIR (30 min ischaemia, 2 h reperfusion). We measured infarct size, markers of cardiac oxidative stress, myocardial phosphorylation state of mitogen-activated protein (MAP) kinases and AKT, expression levels of chemokines and cytokines in the heart and plasma, as well as cardiac function by echography and conductance volumetry. TLR5-deficient mice had normal cardiac morphology and function under physiological conditions. After MIR, the absence of TLR5 promoted an increase in infarct size and myocardial oxidative stress. Lack of TLR5 fostered p38 phosphorylation, reduced AKT phosphorylation and markedly increased the expression of inflammatory cytokines, whereas it precipitated acute LV (left ventricle) dysfunction. Therefore, contrary to the detrimental roles of TLR2, TLR3 and TLR4 in the infarcted heart, TLR5 is important to limit myocardial damage, inflammation and functional compromise after MIR.
MI triggers an inflammatory response orchestrated by members of the TLRs. Detrimental roles have been reported for TLR2, TLR3 and TLR4 and thus pharmacological inhibitors of these receptors might be helpful to limit inflammatory damage in the infarcted myocardium. Whether other TLRs play a role in this setting is unknown.
In the present study, we found that genetic deficiency of TLR5 significantly aggravates myocardial damage, inflammation and dysfunction after experimental MIR, through mechanisms involving enhanced myocardial oxidative stress and activation of p38 MAP (mitogen-activated protein) kinase.
Therefore, in contrast with the deleterious functions of other TLRs, TLR5 appears protective during MI. As a result, targeting TLR5 by pharmacological activators could have therapeutic interest during acute MI.
Myocardial infarction (MI) elicits a sterile inflammation as a primary response to myocardial injury, which sets-up the steps towards healing of the cardiac damage. Although essential to repair the injured myocardium, this response can also promote various maladaptive effects eventually resulting in adverse ventricular remodelling, functional compromise and clinical heart failure. Understanding the molecular mechanism of post-MI inflammation is therefore crucial to envision novel therapies to prevent or treat adverse cardiac remodelling . Evidence indicates that this response is triggered by endogenous molecules [damage-associated molecular patterns (DAMPs)], passively released by necrotic cardiac cells or damaged extracellular matrix (ECM), which act as danger signals . DAMPs are sensed by pattern-recognition receptors, which primarily belong to the toll-like receptor (TLR) family and whose engagement activates intracellular signalling promoting the expression of multiple inflammatory mediators [2,3].
Ten different TLRs have been identified in humans, which are all expressed by the heart . The role of TLR-dependent signalling in post-MI inflammation has been recently emphasized by a series of studies showing decreased myocardial damage and immune responses as well as reduced adverse remodelling in mice deficient in TLR2 , TLR3 , TLR4  or the TLR adapters MyD88 (myeloid differentiation primary response gene 88)  and Trif (TLR-associated activator of interferon) . Endogenous ligands of TLR2 and TLR4 in this setting may comprise high-mobility group box-1 protein (HMGB1) , fibronectin-EDA (extra domain A)  and heat shock proteins , whereas extracellular RNA has emerged as the ligand of TLR3 . The importance of TLR signalling in post-MI inflammation suggests that other members of the TLR family could also be implicated. We previously reported that the myocardium possesses an intact TLR5 signalling machinery, as evidenced by the activation of prototypical innate immune responses in cardiac tissue exposed to bacterial flagellin, the only known ligand of TLR5, both in vitro and in vivo . Therefore, to explore a possible involvement of TLR5 in the establishment of post-MI inflammation, we compared the response of wild-type (WT) and TLR5-deficient (TLR5−/−) mice to myocardial ischaemia–reperfusion (MIR) injury.
MATERIAL AND METHODS
All experiments in homozygous male TLR5-deficient mice with C57BL/6 background (8–10 weeks old, derived from the original line developed by Akira ) and WT age-matched male C57BL/6 mice (Janvier Labs), were conducted according to national and international guidelines, with the approval of the Local Institutional Animal Care and Use Committee (Service of Veterinary Affairs, State of Vaud, Switzerland, authorization Nr 2484). A supplementary Table (Supplementary Table S1) provides the number of animals used in each of the different experiments.
Myocardial ischaemia-reperfusion and determination of infarct size
Mice were anaesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (10 mg/kg), tracheotomized and ventilated (tidal volume 200 μl, 120 strokes/min, Mouse Ventilator model 687, Harvard Instruments). The heart was exposed by a left thoracotomy at the fourth intercostal space and the left anterior descending (LAD) coronary artery was occluded with a 7.0 suture, 1 mm below the left atrium, according to our previously published procedure . After 30 min ischaemia, the suture was released to allow reperfusion for 2 h (MIR group). A group of mice with the same surgical procedure but without occlusion (Sham group) and a group of mice with no surgery (baseline group), were used for control purposes. At the end of reperfusion, myocardial area at risk (AAR) and infarct size were determined in 12 animals/genotype by standard Evans Blue–triphenyl tetrazolium chloride (TTC) colorimetric method  and quantified by image analysis using the Java image processing program ImageJ, v1.45.
Assessment of left ventricular structure and function
Baseline left ventricle (LV) function was evaluated in WT (n=8) and TLR5−/− (n=7) mice using transthoracic echocardiography (M-mode 2D echocardiography) performed in anaesthetized mice [ketamine/xylazine, 80 mg/kg/5 mg/kg, intraperitoneally (i.p.)] using a Sequoia C256 ultrasound machine with a 15 mHz liner array transducer. LV chamber dimensions [LV end-diastolic dimension (LVEDD); LV end-systolic dimension (LVESD)], LV wall thicknesses (LV posterior and anterior walls), fractional shortening (FS, calculated as [(LVEDD − LVDSD)/LVEDD] × 100%) were analysed offline from M-mode traces, using Philips Xcelera software. LV function was further determined by pressure–volume (PV) technology, under baseline conditions (n=6–8 mice/genotype) and after MIR (n=5 animals/genotype), using a microtip 1F catheter (PVR-1045; Millar Instruments) inserted through the apex of the LV at the end of reperfusion. PV signals were recorded with an MPVS-300 PV conductance system (Millar Instruments). Volume calibration and determination of LV parallel conductance were performed as described . A 5-0 silk suture passed around the inferior vena cava was transiently tied to acutely reduce venous return, to compute the slope [Ees (end-systolic elastance)] and the zero pressure intercept (V0) of successive end-systolic PV relationships (ESPVR), to evaluate contractility. Due to large shifts in V0, we calculated the maximal end-systolic pressure (Pes) reached at a given end-systolic volume (Ves) of 25 μl (Pmax@25 μl), as a corrected index of LV contractility, according to our previous methodological description .
Determination of plasma and myocardial cytokines
At the end of experiments, blood was withdrawn by cardiac puncture, the heart was retrieved, the LV was isolated, cut and pulverized in liquid nitrogen. Myocardial powder was homogenized (Tris/HCl 10 mM, NP40 0.5%, NaCl 0.15 M, Na3VO4 1 mM, NaF 10 mM, PMSF 1 mM, EDTA 1 mM, aprotinin 10 μg/ml, leupeptin 10 μg/ml and pepstatin 1 μg/ml) and proteins were measured by the BCA assay (Thermo Scientific Pierce). The concentrations of interleukin-6 (IL-6) and of the chemokines CXCL2 [chemokine (C-X-C motif) ligand 2]–MIP-2 (macrophage inflammatory protein 2), CCL2 [chemokine (C-C motif) ligand 2]–MCP-1 (monocyte chemoattractant protein 1) and KC (keratinocyte chemoattractant), were determined in baseline, sham and MIR groups (in each group, n=6–7 mice/genotype, except for plasma KC, n=3–7 mice/genotype) using BioPlex™ multiplex cytokine assays or a commercial ELISA (MCP-1 ELISA kit, R&D Systems). Plasma was also assayed for creatine kinase activity (n=6–9 mice/genotype in each group), using a Cobas 8000 automated analyzer (Roche Diagnostics).
Biomarkers of myocardial oxidative stress
Oxidative modifications of myocardial proteins was evaluated by the level of protein–carbonyl adducts (n=5 mice/genotype), using the OxiSelect™ Protein Carbonyl ELISA Kit (Cell Biolabs). Lipid peroxidation was evaluated by the formation of 4-hydroxynonenal (HNE)–histidine adducts in myocardial proteins (n=4–5 mice/genotype), using the OxiSelect™ HNE-His Adduct ELISA Kit (Cell Biolabs), as well as by the formation of malondialdehyde (MDA, n=6–7 mice/genotype), measured as thiobarbituric acid-reactive substances in heart homogenates, as described . The myocardial activity of the antioxidant enzyme catalase was determined in the MIR group (n=5 mice/genotype), by measuring the decomposition of H2O2 followed by spectrophotometry at 240 nm, as reported previously .
SDS/PAGE and Western immunoblotting
Myocardial proteins (20 μg) were separated by standard SDS/PAGE procedure and were electro-transferred on to nitrocellulose membranes. Western immunoblotting (n=4–5 mice/genotype in each group) was done using the following primary antibodies: anti-c-Jun N-terminal kinase (JNK)1/2, anti-extracellular signal-regulated kinase (ERK)1/2, anti-p38, anti-AKT, anti-phospho-JNK1/2, anti-phospho-ERK1/2, anti-phospho-p38, anti-phospho-Akt (Cell Signaling) and anti-α-tubulin (Sigma–Aldrich), followed by incubation with appropriate secondary horseradish peroxidase (HRP)-coupled goat-anti rabbit or mouse antibodies (Thermo Scientific Pierce). The immunoblots were revealed with Immobilon Western chemiluminescent HRP substrate (Millipore). Signals were acquired with a Chemi Doc XRS+ imaging system (Bio-Rad) and densitometric analyses were made with ImageJ software v1.45.
Histology and immunohistology
Hearts from baseline and MIR groups (n=3 mice/genotype) were fixed in 4% paraformaldehyde, embedded in paraffin, cut in 5 μm slices and stained with haematoxylin and eosin. Deparaffinized sections were incubated overnight at 4°C with rat anti-mouse Gr-1/Ly-6G monoclonal antibody (R&D systems, Minneapolis) to detect mature granulocytes, followed by incubation with a rabbit anti-rat biotin-conjugated secondary antibody, as described .
Data are presented as means±S.E.M. of n observations. Experiments comparing only two conditions were analysed by Student's t test. The other experiments were evaluated using the Holm–Sidak method for multiple comparisons. Statistical significance was assigned to P<0.05. All statistics were performed using GraphPad Prism 6 software.
TLR5−/− and WT mice disclose a comparable cardiac phenotype
Comparable cardiac phenotypes in TLR5−/− and WT mice
Lack of TLR5 increases myocardial reperfusion injury and oxidative stress
Whereas AAR was similar in WT and TLR5−/− mice (Figure 2A), infarct size, in percent of the AAR (Figure 2B) or of the LV (Figure 2C), was significantly larger in TLR5-deficient animals. Figure 2(D) shows a representative example of Evans Blue–TTC staining of WT and TLR5−/− hearts. Plasma CK (creatine kinase) activity also increased significantly more in TLR5 knockout (KO) mice (Figure 2E). Of note, a slight increase in plasma CK occurred in sham animals, reflecting some damage to striated muscles, as well as minor cardiac damage related to the surgery. The increased infarct size in TLR5-deficient mice was associated with greater oxidative damage as shown by higher levels of protein carbonyl adducts (Figure 3A), 4-HNE (Figure 3B) and MDA (Figure 3C). In parallel, the antioxidant enzyme catalase displayed a significantly smaller activity in the myocardium of TLR5−/− mice after MIR (Figure 3D).
Infarct size is larger in TLR5−/− mice after MIR
The absence of TLR5 promotes greater oxidative stress after MIR
TLR5 deficiency increases p38 phosphorylation during MIR
Although the phosphorylation of ERK (Figure 4A) and JNK (Figure 4B) increased in both genotypes during MIR, the change was significant only for JNK phosphorylation in WT mice, with no significant differences between genotypes. Alternatively, phosphorylation of p38 during MIR was significantly greater in TLR5−/− mice (Figure 4C), whereas phosphorylation of AKT was significantly greater in WT animals (Figure 4D).
TLR5 deficiency elicits distinct regulations of p38 and AKT during MIR
TLR5 deficiency promotes cardiac and systemic innate immune responses during myocardial infarction
Cardiac chemokines and cytokines were barely detectable under basal conditions and after sham surgery in WT and TLR5−/− mice (Figure 5). In contrast, the innate immune response triggered by MIR was characterized by increased expressions of CXCL2 (Figure 5A), IL-6 (Figure 5B) and CCL-2 (Figure 5C), which were significantly greater in TLR5−/− animals. Histochemical detection of GR-1 positive cells indicated the presence of infiltrating mature granulocytes in comparable amounts in WT and TLR5−/− mice (Figure 5D).
Increased immune responses in the heart in the absence of TLR5 during MIR
In keeping with the findings in the myocardium, plasma levels of cytokines were very low under baseline conditions (Figure 6). Sham surgery triggered a slight increase in CXCL2 (Figure 6A), KC (another CXC chemokine; Figure 6B) and IL-6 (Figure 6C) in both genotypes. These increases were significantly more pronounced following MIR, except for CCL2, which only increased in TLR5−/− mice (Figure 6D). (Of note, plasma CCL2 was not measured in sham groups due to a technical problem). During MIR, levels of CXCL2, KC and CCL2 were significantly higher in TLR5−/− mice, whereas IL-6 values were comparable between WT and TLR5−/− mice.
TLR5−/− mice have greater plasma chemokines levels after MIR
Acute LV systolic dysfunction occurs during MIR in the absence of TLR5
Under baseline conditions, LV pressures and volumes, heart rate, ejection fraction (EF) and cardiac output were comparable between WT and TLR5−/− mice (Figure 7). After MIR, cardiac function remained preserved in WT animals, whereas there was a trend towards cardiac dilation with significant reduction in stroke volume in TLR5−/− mice, responsible for a significant reduction in EF (Figure 7G). Figures 7(I) and 7(J) show examples of PV relationships in WT and TLR5−/− mice under baseline and MIR conditions. We then computed the slope of ESPVR (Ees) and its V0 at rapidly changing preload to evaluate LV contractility . Although we did not notice any significant differences with respect to Ees (Figure 8A), there was a significant shift to the right of V0 in TLR5−/− mice during MIR (Figure 8B). Since such a right shift of V0 is indicative of reduced LV contractility, we determined the maximal Pes at a given Ves of 25 μl (Pmax at 25 μl) as a corrected index of LV contractility . This index was significantly smaller in TLR5−/− mice (Figure 8C), pointing to depressed LV inotropism in these animals. Illustrative examples of ESPVR relationships are shown in Figures 8(D) and 8(E).
Lack of TLR5 alters the haemodynamic response to MIR
MIR is associated with reduced LV contractility in the absence of TLR5
The results of our study indicate that the genetic absence of TLR5 significantly exacerbates the consequences of MIR. TLR5-deficient mice exhibited larger myocardial infarcts, greater oxidative stress and enhanced innate immune responses than WT animals and the lack of TLR5 fostered the development of LV systolic dysfunction in this setting.
MI elicits a steady inflammatory response essential for proper healing of the myocardial injury, but which may also set in motion pathological processes contributing to the pathogenesis of adverse remodelling . A key role of TLR signalling in such responses has been recently established, as indicated by decreased infarct size, reduced inflammatory response and improved functional status in mice with targeted disruption of TLR2 , TLR3 , TLR4  and the TLR adapters MyD88  and Trif . Therefore, TLRs have generally been viewed as responsible for deleterious inflammatory cascades in the infarcted heart, which suggests that anti-TLRs strategies might prove useful in this setting . Our findings challenge this concept, as they support, instead, the existence of TLR5-dependent protective mechanisms during MIR, which were independent from any fundamental phenotypic variation between WT and TLR5−/− animals.
An important consequence of the lack of TLR5 was the promotion of myocardial oxidative stress, as shown by increased formation of carbonyl protein adducts and products of lipid peroxidation, together with reduced catalase activity after MIR. The latter account for 80% of all peroxidase activity in cardiomyocytes and its ability to detoxify H2O2 is an essential safeguarding process in MI . MIR increases catalase activity as an important adaptive process , via mechanisms involving AKT-dependent activation of FoxO (forkhead box O) transcription factors [22,23]. AKT is a crucial survival kinase activated as a protective mechanism, notably during ischaemia-reperfusion . Such activation was indeed present in the reperfused heart from WT, but not TLR5−/− mice, implying that TLR5 is implicated in the establishment of this cardinal cardioprotective pathway. This would be consistent with previous data in intestinal cells, showing that TLR5 promotes AKT phosphorylation as a negative feedback mechanism to prevent excessive immune responses .
A second major finding in TLR5−/− mice was the significant myocardial phosphorylation of p38 and the substantial increase in myocardial cytokines/chemokines upon reperfusion. Activated p38 is a key pathway promoting cardiomyocyte death, as identified from studies using genetic or pharmacological suppression of p38 signalling [26,27]. As such, p38 may have largely contributed to the increased myocardial injury in TLR5−/− mice. Free radicals and oxidants have been identified as crucial activators of p38, notably in cardiomyocytes , supporting the contention that the prominent myocardial oxidative stress in TLR5−/− mice was the most likely trigger of p38 activation. Besides its cell-death promoting actions, p38 also conveys significant pro-inflammatory signals, as identified by adenovirus-mediated gene transfer of p38 in rat myocardium . Rats overexpressing p38 exhibited increased expression of multiple inflammatory genes, primarily those encoding IL-6 and CCL2, i.e. precisely two proteins markedly increased in the heart of TLR5−/− mice. Therefore, we propose that myocardial p38 activation was responsible, at least partly, for such increased expression. Importantly, both CCL2 and IL-6 have major prognostic value in MI in humans [30,31]. Our findings suggest, therefore that deficient TLR5 signalling could be mechanistically involved in the up-regulation of these inflammatory cytokines in the clinical setting.
Besides IL-6 and CCL2, TLR5−/− mice displayed a significant increase of cardiac CXCL2, a murine equivalent of human IL-8 with neutrophil chemoattractant properties. CXCL2 has been reported to be induced in cardiac venules after brief periods of reperfusion (3 h), through a reactive oxygen species-dependent mechanism , which can be also retained here, owing to the marked oxidative stress in TLR5−/− mice. In spite of these dissimilarities in chemokine expression, we did not notice obvious differences in PMN (polymorphonuclear) infiltration between WT and TLR5−/− mice. This could be attributed to the short reperfusion time in our study (2 h), at a moment when PMN infiltration is far from its peak, which occurs between 6 and 24 h after myocardial reperfusion .
MIR further induced a state of systemic inflammation, indicated by increased plasma levels of IL-6, CCL2, CXCL2 and KC, another CXC chemokine related to human IL-8. In the acute phase of MI, the increase in plasma cytokines mostly reflects a spill-over of locally produced cytokines within the injured myocardium . Thus, it was not surprising that the profile of plasma chemokines was similar to that of the myocardium between TLR5−/− and WT mice, with significantly higher levels in TLR5-deficient animals. However, this was not the case for IL-6, since, in contrast with the heart, its systemic increase was similar among both genotypes. We speculate that extra-cardiac sources of IL-6, especially peripheral blood mononuclear cells (PBMCs), contributed to its systemic increase, via mechanisms independent from TLR5. This would be entirely consistent with recent data showing that during MI, PBMCs produce IL-6 in response to circulating cytokines  and plasma IL-6 levels are unrelated to the extent of myocardial necrosis .
The increased myocardial damage and inflammatory response in TLR5−/− mice was associated with significant functional impairment, as evidenced by reduced EF and contractility. Interestingly, such impairment was not present in WT mice, indicating that the hyperacute model used in the present study (2 h) does not produce overt LV dysfunction in WT mice. The reduction in EF in TLR5−/− mice occurred in spite of a slight increase in preload [increased end-diastolic volume (Ved)] and no apparent change in afterload (similar Pes), implying reduced contractility as the primary causal mechanism. Depressed LV inotropy was confirmed by the significant reduction in LV systolic pressure at a given Ves (Pmax at 25 μl). This index was used to take into account the fact that V0, the zero-pressure intercept of the ESPVR, significantly shifted to the right after MIR in TLR5−/− mice, which invalidates the concept of linear elastance (Ees) to evaluate contractility [16,36]. Multiple abnormalities noted in the present study may have contributed to impair LV function. These include oxidative stress, which depresses cardiac contractility through innumerable mechanisms , activation of p38, which exerts negative inotropic effects via the phosphorylation of myofilament proteins or of Ca2+ handling proteins  and inflammatory cytokines, especially IL-6, which has well-known negative effects on contractility .
Our study has certain limitations. First, we used C57BL/6 mice as WT controls, but not TLR5+/+ littermates, which may have introduced some biases in our observations. Secondly, we evaluated the response of TLR5−/− mice only in an acute model of MIR. The possible long-term consequences, notably in terms of LV remodelling and chronic heart failure, can therefore not be anticipated from the current data. However, given that the acute inflammatory changes occurring after MIR are directly correlated with prognosis , we hypothesize that the hyper-inflammation observed in TLR5−/− mice should translate into impaired long-term morphological and functional consequences, which should be evaluated in future studies. Thirdly, we cannot be precise as to whether cardiac or extracardiac (primarily leucocytes) deficiency of TLR5 was responsible for the negative outcome of TLR5 deficiency. However, the marked differences in myocardial integrity, biology and function between WT and TLR5-deficient animals occurred at a time when cardiac infiltration by circulating leucocytes was not significantly different. This argues against a major role of TLR5 deficiency in cells of non-cardiac origin in the observed results.
In conclusion, our study indicates that the genetic deficiency of TLR5 markedly enhances the detrimental consequences of MI, as indicated by larger infarct size, greater cardiac and systemic expression of inflammatory cytokines/chemokines, as well as more severe LV dysfunction. These effects are associated with significant myocardial oxidative stress and p38 phosphorylation, together with reduced AKT phosphorylation. Our results contrast with the injurious actions of TLR signalling in the infarcted heart reported for TLR2, TLR3 and TLR4, as they suggest instead that TLR5 conveys cardioprotective signals in this setting. Thus, the development of pharmacological activators of TLR5 might prove useful to improve the outcome of MI.
Roumen Parapanov, Jérôme Lugrin, Giuseppina Milano, Catherine Vergely and Na Li performed the experiments. François Feihl, Nathalie Rosenblatt-Velin and Bernard Waeber provided reagents and contributed conceptually to the project. Lucas Liaudet supervised the project and obtained funding. Lucas Liaudet and Pal Pacher wrote the manuscript.
This work was supported by the Swiss National Foundation for Scientific Research [grant numbers 310030_135394/1 (to L.L.) and 310030_132491 (to N.R.-V.)]; the Emma Muschamp Foundation; the Mahmoud Darvish Foundation; and the Intramural Program of National Institutes of Health/NIAAA (to P.P.).
area at risk
chemokine (C-C motif) ligand 2
chemokine (C-X-C motif) ligand 2
damage-associated molecular pattern
extracellular signal-regulated kinase
end-systolic pressure-volume relationship
c-Jun N-terminal kinase
left ventricle end-diastolic dimension
- MAP kinase
mitogen-activated protein kinase
monocyte chemoattractant protein 1
myeloid differentiation primary response gene 88
peripheral blood mononuclear cell
toll-like receptor-associated activator of interferon
triphenyl tetrazolium chloride
zero pressure intercept