11.4D: Toll-Like Receptors - Biology

11.4D: Toll-Like Receptors - Biology

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Learning Objectives

  • Summarize Toll-like receptors

Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system as well as the digestive system. They are single, membrane-spanning, non-catalytic receptors that recognize structurally conserved molecules derived from microbes. Once these microbes have breached physical barriers such as the skin or intestinal tract mucosa, they are recognized by TLRs, which activate immune cell responses.

TLRs are a type of pattern recognition receptor (PRR) and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs). TLRs together with the Interleukin-1 receptors form a receptor superfamily, known as the “Interleukin-1 Receptor/Toll-Like Receptor Superfamily”; all members of this family have in common a so-called TIR (Toll-IL-1 receptor) domain.

Because of the specificity of Toll-like receptors (and other innate immune receptors) they cannot easily be changed in the course of evolution, these receptors recognize molecules that are constantly associated with threats (i.e., pathogen or cell stress) and are highly specific to these threats (i.e., cannot be mistaken for self molecules). Pathogen-associated molecules that meet this requirement are usually critical to the pathogen’s function and cannot be eliminated or changed through mutation; they are said to be evolutionarily conserved. Well-conserved features in pathogens include bacterial cell-surface lipopolysaccharides (LPS), lipoproteins, lipopeptides, and lipoarabinomannan; proteins such as flagellin from bacterial flagella; double-stranded RNA of viruses; or the unmethylated CpG islands of bacterial and viral DNA; and certain other RNA and DNA. For most of the TLRs, ligand recognition specificity has now been established by gene targeting (also known as “gene knockout”): a technique by which individual genes may be selectively deleted in mice. See the table below for a summary of known TLR ligands.

TLRs are believed to function as dimers. Though most TLRs appear to function as homodimers, TLR2 forms heterodimers with TLR1 or TLR6, each dimer having a different ligand specificity. TLRs may also depend on other co-receptors for full ligand sensitivity, such as in the case of TLR4’s recognition of LPS, which requires MD-2. CD14 and LPS-Binding Protein (LBP) are known to facilitate the presentation of LPS to MD-2.

The adapter proteins and kinases that mediate TLR signaling have also been targeted. In addition, random germline mutagenesis with ENU has been used to decipher the TLR signaling pathways. When activated, TLRs recruit adapter molecules within the cytoplasm of cells in order to propagate a signal. Four adapter molecules are known to be involved in signaling. These proteins are known as MyD88, Tirap (also called Mal), Trif, and Tram.

TLR signaling is divided into two distinct signaling pathways, the MyD88-dependent and TRIF-dependent pathway. The MyD88-dependent response occurs on dimerization of the TLR receptor, and is utilized by every TLR except TLR3. Its primary effect is activation of NFκB. Ligand binding and conformational change that occurs in the receptor recruits the adaptor protein MyD88, a member of the TIR family. MyD88 then recruits IRAK 4, IRAK1 and IRAK2. IRAK kinases then phosphorylate and activate the protein TRAF6, which in turn polyubiquinates the protein TAK1, as well as itself in order to facilitate binding to IKKβ. On binding, TAK1 phosphorylates IKKβ, which then phosphorylates IκB causing its degradation and allowing NFκB to diffuse into the cell nucleus and activate transcription.

Both TRL3 and TRL4 utilize the TRIF-dependent pathway, which is triggered by dsRNA and LPS, respectively. For TRL3, dsRNA leads to activation of the receptor, recruiting the adaptor TRIF. TRIF activates the kinases TBK1 and RIP1, which creates a branch in the signaling pathway. The TRIF/TBK1 signaling complex phosphorylates IRF3 allowing its translocation into the nucleus and production of Type I interferons. Meanwhile, activation of RIP1 causes the polyubiquination and activation of TAK1 and NFκB transcription in the same manner as the MyD88-dependent pathway.

TLR signaling ultimately leads to the induction or suppression of genes that orchestrate the inflammatory response. In all, thousands of genes are activated by TLR signaling, and collectively, the TLRs constitute one of the most pleiotropic yet tightly regulated gateways for gene modulation.

Toll-like receptors bind and become activated by different ligands, which, in turn, are located on different types of organisms or structures. They also have different adapters to respond to activation and are located sometimes at the cell surface and sometimes to internal cell compartments.

Key Points

  • TLRs are a type of pattern recognition receptor (PRR).
  • TLRs recognize molecules that are broadly shared by pathogens but distinguishable from host molecules, collectively referred to as pathogen-associated molecular patterns (PAMPs).
  • TLR signaling is divided into two distinct signaling pathways, the MyD88-dependent and TRIF-dependent pathway.

Key Terms

  • Toll-like receptor: Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system as well as the digestive system. They are single, membrane-spanning, non-catalytic receptors that recognize structurally conserved molecules derived from microbes.
  • innate immune system: This is the initial line of defense that entails a cascade of cells and mechanisms that protect the host from infection by different organisms in an indeterminate pattern.
  • signaling pathway: Signal pathways occurs when an extracellular signaling molecule activates a cell surface receptor. In turn, this receptor alters intracellular molecules creating a response. There are two stages in this process:A signaling molecule activates a specific receptor protein on the cell membrane.A second messenger transmits the signal into the cell, eliciting a physiological response.In either step, the signal can be amplified. Thus, one signaling molecule can cause many responses.

11.4D: Toll-Like Receptors - Biology

Toll-like receptor 4 is a protein that in humans is encoded by the TLR4 gene. TLR4 is a transmembrane protein, member of the toll-like receptor family, which belongs to the pattern recognition receptor (PRR) family. Its activation leads to an intracellular signaling pathway NF-κB and inflammatory cytokine production which is responsible for activating the innate immune system. [4]

TRL4 expressing cells are myeloid (erythrocytes, granulocytes, macrophages) rather than lymphoid (T-cells, B-cells, NK cells). [4] Most myeloid cells also express high levels of CD14, which facilitates activation of TLR4 by LPS. [5]

It is most well known for recognizing lipopolysaccharide (LPS), a component present in many Gram-negative bacteria (e.g. Neisseria spp.) and selected Gram-positive bacteria. Its ligands also include several viral proteins, polysaccharide, and a variety of endogenous proteins such as low-density lipoprotein, beta-defensins, and heat shock protein. [6] Palmitic acid is also a TLR4 agonist. [7]

TLR4 has also been designated as CD284 (cluster of differentiation 284). The molecular weight of TLR4 is approximately 95 kDa.

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Toll-Like Receptor Signaling Inhibits Hepatitis B Virus Replication In Vivo

FIG. 1 . Toll-like receptor ligands inhibit HBV replication in vivo. Age-, sex-, and serum HBeAg-matched lineage 1.3.32 HBV transgenic mice were injected intravenously with 20 μg (not shown) and 100 μg of TLR2/6 (peptidoglycan [PGN] from Staphylococcus aureus InvivoGen), TLR2/1 ligand N-α-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]- l -cysteine (Pam3Cys InvivoGen), 10 μg of TLR3 [polyinosinic-poly(I · C) P(I:C) Sigma]) TLR4 (LPS from Escherichia coli 011:B4 strain InvivoGen), TLR5 (Flagellin from salmonella muenchen Calbiochem), TLR7 (R848) (InvivoGen), or TLR9 (ODN 1826 CpG oligonucleotide [CpG] InvivoGen) ligands and sacrificed 24 h later. Lineage 1.3.32 mice replicate HBV at high levels in the liver without any evidence of cytopathology (9). (A) Total hepatic DNA was isolated from frozen liver tissues and analyzed for HBV DNA by Southern blot analysis as previously described (9). Bands corresponding to the integrated transgene (Tg), relaxed circular double stranded (RC), and single-stranded (SS) HBV DNA replicative forms are indicated. The integrated transgene can be used to normalize the amount of DNA bound to the membrane. The mean serum ALT activity, measured at the time of autopsy, is indicated for each group and is expressed in units/liter. (B) Total hepatic RNA was also isolated from the same mice and analyzed by RNase protection assay for the expression of various cytokines as previously described (8). The RNA encoding the ribosomal protein L32 was used to normalize the amount of RNA loaded in each lane. FIG. 2 . The antiviral effect of TLRs is mediated by IFN-α/β. Age-, sex-, and serum HBeAg-matched transgenic mice (lineage 1.3.46) that were homozygous (−/−) for the IFN-α/β receptor null mutation (21, 22) were injected with 20 μg of TLRs as indicated and sacrificed 24 h later, and their livers were analyzed for HBV replication. Age-, HBeAg-, and sex-matched transgenic mice from lineage 1.3.46 that were heterozygous (+/−) for the IFN-α/β receptor null mutation or [in the case of poly(I · C) and flagellin] from wild-type lineage 1.3.32 were treated exactly the same as controls. The integrated transgene (Tg) can be used to normalize the amount of DNA bound to the membrane. P(I:C), poly(I · C). FIG. 3 . Freshly isolated hepatocytes do not express TLRs. Primary hepatocytes and intrahepatic lymphocytes were isolated from HBV transgenic mice as previously described (16, 25) and stained with antibodies specific for TLR2, -3, and -9 (eBioscience) in combination with anti-CD11c (BD Pharmingen). Surface staining was performed to detect TLR2 expression, while intracellular staining was applied for TLR3 and -9 expressions (18). Cells were acquired using a FACSCalibur flow cytometer (Becton Dickinson), and data were analyzed using CELLQuest software (Becton Dickinson). Histograms represent TLR staining (gray) or isotype control staining (white) of hepatocytes (left panel). Corresponding TLR expression on CD11c+ cells of intrahepatic lymphocytes were shown as positive controls (right panel).


Toll like receptors (TLRs) are a family of pattern recognition receptors that play a central role in pathogen recognition and shaping the innate immune response. While most of the studies of the role of TLRs have focused on mature immune cell populations, recent reports suggest that TLR signaling may regulate the immune response from the level of the hematopoietic stem cell (HSC). In this study, we sought to further elucidate the effects of systemic TLR ligand exposure on HSCs and determine the cell-intrinsic versus extrinsic effects of such exposure. We specifically focused on TLR2 signaling, as although TLR2 is expressed on HSCs, it’s role in their regulation is not clear. Furthermore, enhanced TLR2 signaling is associated with myelodysplastic syndrome (Wei et al, Leukemia 2013), suggesting that aberrant signaling through this receptor may have clinically significant effects on HSC function.

To elucidate the role of TLR2 signaling in regulating HSCs, we used mice with genetic loss of TLR2, as well as a synthetic agonist of TLR2 (PAM3CSK4) to determine the effects of TLR2 signaling loss or gain, respectively, on HSC cycling, mobilization and function. While TLR2 expression is not required for normal HSC function, treatment of wild-type mice with PAM3CSK4 leads to expansion of HSCs in the bone marrow and spleen, increased HSC cycling, and loss of HSC function in competitive bone marrow transplantation experiments. As TLR2 is expressed on a variety of stromal and hematopoietic cell types, we used bone marrow chimeras (Tlr2 -/- + Tlr2 +/+ marrow transplanted into Tlr2 +/+ recipients) to determine if the effects of PAM3CSK4 treatment are cell intrinsic or extrinsic. The data suggests that HSC cycling and expansion in the marrow and spleen upon PAM3CSK4 treatment are extrinsic (occurring in both transplanted HSC populations), and are associated with increased serum levels of G-CSF. Indeed, inhibition of G-CSF using either a neutralizing antibody or mice lacking the G-CSF receptor (Csf3r -/- ) leads to even further enhanced HSC bone marrow expansion upon G-CSF treatment but significantly reduced numbers of spleen HSCs compared to similarly treated wild-type mice. This suggests mobilization in response to TLR2 signaling is an indirect, G-CSF-mediated process. Ongoing studies are aimed at determining the contribution of G-CSF to the PAM3CSK4- induced loss of HSC function, and determining the source (stromal vs hematopoietic) of G-CSF production upon PAM3CSK4 exposure. Collectively, this data suggest that TLR2 signaling affects HSCs in a largely extrinsic fashion, with G-CSF playing a major role in regulating the effects of TLR2 ligand exposure on HSCs.

Citation (search report)

  • [XD] TAKADA E ET AL: "C-terminal LRRs of human Toll-like receptor 3 control receptor dimerization and signal transmission", MOLECULAR IMMUNOLOGY, PERGAMON, GB, vol. 44, no. 15, 1 July 2007 (2007-07-01), pages 3633 - 3640, XP025320882, ISSN: 0161-5890, [retrieved on 20070530], DOI: 10.1016/J.MOLIMM.2007.04.021
  • [AD] EUNJEONG YANG ET AL: "Cloning of TLR3 isoform.", YONSEI MEDICAL JOURNAL, vol. 45, no. 2, 1 April 2004 (2004-04-01), pages 359 - 361, XP055022273, ISSN: 0513-5796
  • [I] RANJITH-KUMAR C T ET AL: "Biochemical and functional analyses of the human Toll-like receptor 3 ectodomain", JOURNAL OF BIOLOGICAL CHEMISTRY 20070302 AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY INC. US,, vol. 282, no. 10, 2 March 2007 (2007-03-02), pages 7668 - 7678, XP007914953
  • See also references of WO 2010040054A2


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P53 Regulates Toll-Like Receptor 3 Expression and Function in Human Epithelial Cell Lines

FIG. 1 . p53 positively regulates the expression of TLR3 in epithelial cells. (A) HCT116 p53 +/+ and p53 −/− cells were transfected with IL-8 promoter plasmid and treated with 10 μg/ml each of the indicated TLR agonists for 6 h. IL-8 promoter activity was measured 48 h after transfection of IL-8 promoter. The data shown are means ± standard errors from triplicate platings and represent three independent experiments. **, P < 0.001 versus control (Con phosphate-buffered saline [HCT116 p53 +/+ ]) assessed by ANOVA with Dunnett's procedure. (B) TLR3 mRNA in HCT116 p53 +/+ and p53 −/− cells was determined by real-time quantitative PCR. The TLR3 mRNA level was normalized to GAPDH (internal control). The data shown are means ± standard deviations from triplicate determinations from three independent experiments. *, P < 0.05 assessed by Student's t test. (C) TLR3 mRNA was examined in HCT116 p53 +/+ and in HCT116 p53 −/− cells untransfected or transfected with increasing amounts of p53 expression plasmid. (D) TLR3 mRNA was determined in HCT116 p53 +/+ and p53 −/− cells treated for 24 h with 4 or 8 μM 5-FU. For panels C and D, p21 served as a positive control. GAPDH was used as an internal control for RT-PCR analyses. (E) HCT116 p53 +/+ cells were transfected with p53 siRNA (si-p53) or control siRNA duplex (si-GL2), and total RNA from these cells was isolated for analysis of TLR3 and p53 mRNA by RT-PCR. (F) The TLR3 protein level was determined in lysates of HCT116 p53 +/+ and p53 −/− cells by immunoprecipitation (IP) and Western blotting using anti-TLR3 antibody. Hsc70 was used as internal control. con-IgG, control IgG. (G) A549 cells were treated with 4 or 8 μM 5-FU for 24 h. (H) si-GL2 or the indicated amount of p53 siRNA duplex was transfected in A549 cells. Twenty-four hours later, RNA was extracted. (I to K) HepG2 (I), Caco2 (J), and Calu-3 (K) cells were treated with 400 or 800 μM 5-FU for 8 h. For panels G to K, total RNA was extracted and analyzed by RT-PCR for the expression of TLR3, p21 (positive control), or p53. GAPDH served as the internal control. FIG. 2 . p53 binds to and transactivates the TLR3 promoter. (A) HCT116 p53 +/+ and p53 −/− cells were transiently transfected with the indicated TLR3 promoter constructs (0.2 μg). Luciferase activity was determined 48 h after transfection of plasmids and is expressed as activation (fold) over that of the pGL3b vector. Values are means ± standard errors from triplicate platings. The data shown are representative of three independent experiments. ** and ***, P < 0.001 and P < 0.0001, respectively, against the corresponding promoter length in HCT116 p53 −/− cells, as determined by Student's t test. (Right panel) Schematic diagram of the TLR3 promoter constructs containing the p53 binding sites. (B) HCT116 p53 −/− cells were transiently transfected with the indicated TLR3 promoter constructs (0.2 μg) and 0.025 μg p53 plasmid or pcDNA3.1 empty vector (con), and luciferase activity was assayed 48 h posttransfection. Values are means ± standard errors from triplicate platings. Data represent three independent experiments. **, P < 0.001, as determined by Student's t test. (C) HCT116 p53 −/− cells were transiently transfected with pGL3b vector or −2 kb TLR3 promoter (TLR3p), cotransfected with p53 expression plasmid at increasing amounts (6.25, 12.5, and 25 ng), and assayed for luciferase activity. Values are means ± standard errors from triplicate platings. The data shown are representative of three independent experiments. ***, P < 0.0001 versus TLR3p, as assessed by ANOVA with Dunnett's procedure. (D) HCT116 p53 +/+ cells were transiently transfected with the −2 kb TLR3 promoter and cotransfected with si-GL2 or si-p53 (50 or 100 nM) oligonucleotide, and luciferase activity was assayed 48 h posttransfection. Values are means ± standard errors from triplicate platings. The data represent three independent experiments. ** and ***, P < 0.001 and P < 0.0001, respectively, against TLR3p, as determined by ANOVA with Dunnett's test. (E) Wild-type or mutant (mutated p53-binding sites) TLR3 promoter (−2 kb) was transfected into HCT116 p53 +/+ cells, and luciferase activity was assayed 48 h after transfection. Values are means ± standard errors from triplicate platings. Data represent three independent experiments. ***, P < 0.0001 against the wild-type (WT) TLR3 promoter, as determined by ANOVA with Tukey-Kramer's test. n.s., not significant. (Upper panel) Schematic diagram of the −2 kb TLR3 promoter with the indicated position of the mutated p53 binding site. (F) HCT116 p53 −/− cells were transfected with pGL3b and wild-type (WT) or mutant TLR3 promoter and cotransfected with pCDNA3.1 empty vector or p53 plasmid, and luciferase activity was assessed 48 h after transfection. Values are means ± standard errors from triplicate platings. The data represent three independent experiments. * and **, P < 0.01 and P < 0.001, respectively, against pCDNA3.1 vector, as assessed by Student's t test. (G) Consensus site of p53 and the sequence of p53 binding site in the TLR3 promoter at −1929. Underlined bases denote sequence variation from the consensus p53 element. (H) Representative result of the ChIP analysis in HCT116 p53 +/+ cells using p53 antibody or mouse IgG for immunoprecipitation (IP) and primers of the indicated promoter region for PCR. FIG. 3 . Activation of TLR3 signaling pathway by poly(I-C) is impaired in HCT116 p53 −/− cells. (A to C) Cytoplasmic lysates (A and C) or nuclear extracts (B) from HCT116 p53 +/+ and p53 −/− cells unstimulated or stimulated for 3 h with poly(I-C) at the indicated concentration (A and C) or 5 μg/ml (B) were analyzed by Western blotting for phosphorylated IκB-α (p-IκB-α) and basal IκB-α (A), p65 (B), phosphorylated IRF-3 (p-IRF-3), and basal IRF-3 (C) expression. Hsc70 was used as loading control for panels A and C. γ-Tubulin was used as the loading control for panel B. FIG. 4 . The response of cytokines IL-8 and IFN-β, which are downstream of TLR3, to poly(I-C) stimulation requires p53. (A) Real-time quantitative PCR analysis of IL-8 and IFN-β mRNA was performed on HCT116 p53 +/+ and p53 −/− cells untreated or treated with 5 μg/ml poly(I-C) for 3 h. mRNA expression was normalized to GAPDH. Values are means ± standard deviations of triplicate measurements. ***, P < 0.0001, as analyzed by ANOVA with Tukey-Kramer's test. (B) The mRNA level of IL-8 and IFN-β was determined in HCT116 p53 +/+ and p53 −/− cells stimulated with 5 μg/ml poly(I-C) for the indicated time. p21 and GAPDH served as the positive control and internal control, respectively. (C and D) Promoter activity of IL-8 (C) and IFN-β (D) was examined in HCT116 p53 +/+ and p53 −/− cells stimulated with poly(I-C) at the indicated concentration for 6 h (upper panels) or with 5 μg/ml poly(I-C) for the indicated time (lower panels). Values are means ± standard errors from triplicate platings. The data shown are representative of two to three independent experiments. (E) The mRNA expression of IL-8 and IFN-β was analyzed in HCT116 p53 +/+ cells unstimulated or stimulated with poly(I-C) and untransfected or transfected with siRNA duplex for TLR3 (si-TLR3) or p53 (si-p53) or with si-GL2 (for control). Expression of TLR3 and p53 was knocked down by the transfection of their respective siRNAs in p53 +/+ cells (lower panels). FIG. 5 . The TLR3 mRNA level is downregulated in tissues of p53 −/− mice. (A to C) Total RNA isolated from liver (A), intestine (B), and spleen (C) of p53 +/+ , p53 +/− , and p53 −/− mice was analyzed for the expression of TLR3 by real-time quantitative RT-PCR. TLR3 mRNA levels were normalized to HPRT (internal control). The results represent means ± standard deviations (n = 3). * and **, P < 0.01 and P < 0.001, respectively, against p53 wild-type mice (+/+), as assessed by ANOVA with Dunnett's test.

Myocardial Infarction Models: Permanent Coronary Artery Occlusion with Nonreperfused and Reperfused Myocardial Infarction

MI: general considerations.

Coronary occlusion causes immediate cessation of aerobic metabolism in the ischemic myocardium, leading to rapid ATP depletion and metabolite accumulation and resulting in severe systolic dysfunction within seconds (86). If the duration of the ischemic insult is <15 min in larger mammals such as dog and pig, restoration of flow reverses the early ischemic cardiomyocyte changes (transient mitochondrial swelling or glycogen depletion) and all cardiomyocytes in the ischemic area can survive (158). Longer periods of ischemia cause death of an increasing number of cardiomyocytes. A 20- to 30-min interval of severe ischemia is sufficient to induce irreversible changes in some cardiomyocytes of the subendocardial area, inducing sarcolemmal disruption and striking perturbations in mitochondrial architecture, such as ultrastructural evidence of amorphous matrix densities and severe mitochondrial swelling (156). These early ultrastructural alterations mark cardiomyocytes that cannot be salvaged and will ultimately die in the infarct environment (157).

Experimental studies in the canine model of MI demonstrate a transmural heterogeneity in the myocardial response to ischemia, suggesting that the subendocardium, where myocardial oxygen demand is greatest, is more susceptible to ischemic injury than the midmyocardium and subepicardium (2). Thus, the prevailing paradigm suggests a wavefront of cardiomyocyte death that progresses from the more susceptible subendocardium to the less vulnerable subepicardium as the duration of the ischemic insult increases (159, 252). Experimental studies in large animal models have demonstrated that ischemic myocardium cannot be salvaged by reperfusion after 6 h of coronary occlusion (251). The increased vulnerability of subendocardial regions to coronary occlusion may reflect a greater reduction of the subendocardial blood flow due to transmural differences in vascularization (2, 25) and extravascular compression (68, 286). The wavefront concept of ischemia developing into infarction was derived from experimental studies in dogs, where a substantial coronary collateral circulation influences the time course of cardiomyocyte necrosis (86).

The major species difference in the MI response lies in the temporal and spatial kinetics of events and differences due to myocardial size. In mice, durations of coronary occlusion exceeding 60–90 min are considered irreversible, and inflammation and wound healing processes are accelerated (64, 88, 221, 222). In mouse and rat models, reperfused infarcts are typically midmyocardial, and subepicardial and subendocardial regions are relatively spared (50, 69, 325). Studies in a sheep model of reperfused infarction also suggest that the midmyocardium may be most vulnerable to ischemic injury in contrast, the subendocardium is relatively resistant (263). The pig model of coronary occlusion-reperfusion comes closest to human STEMI in its temporal and spatial development, but other models are nevertheless useful to study fundamental mechanisms of MI (140).

MI: technical considerations.

Extensive protocols providing technical details for performing permanent occlusion MI and reperfused MI in mice and rats are available (221, 222, 228, 317, 327). While MI is most commonly performed in rodent models, protocols in other animal models are also available (151, 183, 218, 331). For mice, the quality of open-chest surgery to induce coronary occlusion directly impacts study outcomes (152, 221, 222). Minimizing the size of the thoracotomy and limiting bleeding by entering the thorax through intercostal muscles are recommended.

Biomarkers that have been used to evaluate the presence of MI include cardiac troponins and creatine kinase, and plasma proteins such as macrophage migration inhibitory factor can also be measured as indices of injury (47, 55). Infarct size is widely measured as a key variable for testing genetic or therapeutic intervention efficacy, and infarct size measurements taken serially at both early and late time points can evaluate the extent of infarct expansion (22). Echocardiography can also be used for infarct sizing, with the caveat that echocardiography does not distinguish between reversible ischemic dysfunction (stunning) and irreversible loss of function and therefore a secondary method is needed for confirmation of infarct size at early time points. For more details on measuring cardiac function in mice, the reader is advised to consult the article Guidelines for measuring cardiac physiology in mice (196).

Permanent occlusion MI: model rationale and variables measured.

Permanent coronary occlusion is a relevant animal model of acute STEMI reflective of patients who, due to contraindications or logistic issues, do not receive timely or successful reperfusion (53, 104). Permanent coronary occlusion yields acute ST segment elevation infarction with robust myocardial inflammation and long-term remodeling, thus providing a large effect size that reduces the sample size needed to detect differences between groups. Infarction assessed in the first 1–14 days after coronary ligation is histologically characterized by coagulation band necrosis with a fulminant inflammatory infiltrate in the infarct and border zone regions. Infarction is geometrically and physiologically characterized by wall thinning, increases in LV dimensions and volumes, and decreases in fractional shortening and ejection fraction.

Changes that occur over the first week provide information on myocyte cell death and infarct development, inflammation and leukocyte physiology, extracellular matrix (ECM) turnover and fibroblast activation, and the role of endothelial cells in neovascularization (83, 154, 165, 194, 205). Chronic evaluation at time points 4–8 wk post-MI provides information on long-term remodeling. Whether the infarct region or remote region is the focus of investigation depends on the question asked. Examining the infarct region provides details on active inflammation and scar formation, while examining the remote region provides details on still-viable myocytes within the myocardium and remote inflammatory and ECM processes.

Perioperative and postoperative survival should be assessed, and the time point of delineation between these two phases should be defined. For some laboratories, the perioperative phase includes the time until the animal recovers and becomes ambulatory (usually within 1–3 h for mice). For other laboratories, the perioperative phase includes the first 24 h after surgery. Perioperative death within 24 h post-MI in mice is usually due to surgical errors (or very large infarct sizes), and, in established laboratories, the 24 h surgical mortality rate due to technical issues is <10%. In the permanent occlusion MI model in mice, postoperative death (deaths at >24 h time point) typically occurs during days 3–7 post-MI and is due to rupture, acute heart failure, or arrhythmias (59, 98, 233). Autopsy is strongly recommended for all mice that die prematurely, to evaluate early deaths due to technical issues and later deaths due to complications of MI. Seven-day postoperative mortality rates are

10–25% (75–90% survival) for female young mice and 50–70% (30–50% survival) for male young mice (47, 61, 89, 98, 152, 170, 195, 202, 206, 234, 310–312, 319, 323). Immediate survival from the surgery can also be affected by baseline characteristics such as obesity, diabetes, or high levels of circulating inflammatory cells, which, in turn, determine the response to anesthesia and surgery (60, 123, 202, 203). While there is no difference in infarct tolerance between young and middle-aged mice (323), older mice may survive better than younger mice (202, 319).

For permanent occlusion MI models, infarct size must be measured in fresh LV slices at the time of necropsy by TTC staining and typically ranges from 30% to 60% of the total LV (47, 48, 61, 89, 98, 152, 195, 202, 206, 223, 310–312, 319, 323). The method for calculating infarct size varies across laboratories. Some laboratories use area calculations, other laboratories measure length in the midmyocardium, and other laboratories measure and average lengths in the subendocardium and subepicardium. There is no need to use Evans blue for area at risk assessment in permanent coronary occlusion models that pass the point from ischemia to infarction, as the entire area at risk is infarcted. It is important that MI surgical success is confirmed and that the initial infarct injury is comparable across groups, to assess remodeling differences at later stages. In mice, ligating the coronary artery at the same anatomical location across groups is important 1 mm distal to the left atrium is the recommended site to generate large infarcts (35–60% of total LV). Failure to induce MI can occur, usually due to missing the coronary artery during the ligation step. Monitoring the electrocardiogram for ST segment elevation during the procedure reduces this possibility. Echocardiography at 3 h after coronary occlusion can be used to exclude animals with excessively small or large MI before randomizing groups (153, 155, 195). Late gadolinium-enhanced MRI is also useful for selecting animals with consistent infarct sizes (262). When assessing effects of treatments initiated post-MI, it is important to show that infarct size is not different between groups before treatment. Plasma sampling at 24 h post-MI can be used to assay cardiac biomarkers, such as troponins and inflammatory cytokines, with the caveat that these measurements can indicate presence or absence of infarct and not extent of injury. After coronary occlusion, care should be taken in performing these assessments to minimize disturbing animals at times when cardiac rupture may be triggered by stress, particularly at days 3–7 post-MI in untreated controls (96, 98). Small infarcts may reflect technical issues in missing the coronary artery, resulting in damage from the suture rather than reflecting the intended myocardial ischemia and infarction. Infarct sizes <30% are typically excluded. If included, small and large infarcts may need to be grouped separately to reduce possible type II statistical errors.

Cardiac wound healing and remodeling, typically assessed days to weeks post-MI, can be examined using a wide variety of approaches, including echocardiography, histology, biochemistry, and cell biology (5, 217, 328). Serial measurements of cardiac geometry and function by echocardiography are useful for defining phenotypes. Cardiac dimensions vary depending on heart rate and depth of anesthesia, and these parameters must be carefully controlled and matched across groups. Cardiac functional reserve can be assessed by measuring the contractile response to inotropic drugs or volume overload. Cardiac MRI and hemodynamic assessment by pressure-volume catheterization are other ways to measure cardiac physiology parameters. It is feasible to quantify infarct size noninvasively and serially by using cardiac MRI (181). Hemodynamic evaluation in mice is a terminal procedure, which prevents its use in serial assessments.

Hematoxylin and eosin staining provides information on areas of necrosis and inflammation, while picrosirius red staining provides information on total collagen accumulation both in the scar and remote regions (316). Immunohistochemistry for neutrophils, macrophages, lymphocytes, fibroblasts, and endothelial cells provides information on the extent of inflammation, scar formation, and neovascularization. Isolating individual cell types and assessment of ex vivo phenotypes in culture can further aid in understanding mechanisms. Studies have revealed that inflammation evoked by acute myocardial infarction also occurs systemically and that the spleen and liver are important sources of cells and factors that influence LV remodeling (71, 72, 95, 116, 198, 199, 293).

I/R MI: model rationale and variables measured.

Implementation of myocardial reperfusion strategies has significantly reduced mortality in acute STEMI. Reperfusion has contributed to the growing pool of patients who survive the acute event and are at risk for adverse remodeling and subsequent development of heart failure (133, 136). In addition to salvaging cardiomyocytes, reperfusion has profound effects on cellular events responsible for repair and remodeling.

Although timely reperfusion is essential to salvage viable cardiomyocytes from ischemic death, extensive preclinical and clinical evidence suggests that reperfusion itself causes injury (119, 121, 147). Reperfusion-induced arrhythmias and myocardial stunning are self-limited and reversible forms of reperfusion injury, while microvascular obstruction and lethal cardiomyocyte injury are irreversible and extend damage, thus contributing to adverse outcomes following MI (13, 126, 177, 241, 318). In patients, no reflow during reperfusion may be exacerbated due to the generation of microemboli composed of atherosclerotic debris and thrombi during percutaneous coronary interventions (135, 253).

MI both with or without reperfusion shares many of the same technical guidelines, and this information is provided above. The one technical difference is whether the ligation is removed at 45–60 min after the occlusion to reperfuse the myocardium. Similar to permanent occlusion MI, studies investigating the inflammatory and reparative response following MI with reperfusion need to take into account the dynamic sequence of cellular events involved in repair. Common measurements shared by the two MI models are shown in Table 2. For studies aimed at investigating acute myocardial injury using a reperfusion strategy, the duration of coronary occlusion needs to be sufficient for the induction of significant MI but not overly prolonged to cause irreversible injury in the entire area at risk. From the cell physiology perspective, the reparative response after MI can be divided into three distinct but overlapping phases: inflammation, proliferation, and maturation (26, 65). In the infarcted myocardium, dying cardiomyocytes release damage-associated molecular patterns and induce cytokines and chemokines to recruit leukocytes into the infarcted region, thus triggering an intense inflammatory reaction that serves to clear the infarct from dead cells and ECM debris, while initiating a reparative response (84). Early reperfusion after irreversible cardiomyocyte injury accelerates and accentuates the inflammatory reaction and has profound effects on the pathological features of the infarct. Microvascular hyperpermeability is evident in the myocardium with acute I/R (97). Rapid extravasation of blood cells through the hyperpermeable vessels may result in hemorrhagic changes (98, 178). Influx of phagocytotic macrophages is accelerated, resulting in more rapid removal of dead cardiomyocytes compared with permanent occlusion MI. In reperfused infarcts, dying cardiomyocytes often exhibit large contraction bands, comprised of hypercontracted sarcomeres. Subsarcolemmal blebs and granular mitochondrial densities, which are already present in irreversibly injured cardiomyocytes before restoration of blood flow, become more prominent upon reperfusion.

Table 2. Common output measurements for in vivo MI and MI/reperfusion studies

MI, myocardial infarction ECM, extracellular matrix MRI, magnetic resonance imaging.

Phagocytosis of dead cells by activated macrophages results in the activation of endogenous anti-inflammatory pathways, ultimately leading to resolution of the inflammatory infiltrate. Suppression of inflammation is followed by recruitment of activated myofibroblasts that deposit large amounts of ECM proteins and by activation of angiogenesis (145). As the scar matures, fibroblasts become quiescent and infarct neovessels acquire a coat of mural cells (332). Compared with large mammals, rodents exhibit an accelerated time course of infiltration with inflammatory and reparative cells (64).

Leukocyte infiltration during the inflammatory phase of infarct healing and myofibroblast activation and accumulation during the proliferative phase are predominantly localized in the infarct region and border zone (87, 122, 280). During scar maturation, the cellular content in the infarcted region is reduced. At the same time, however, the number of activated macrophages and fibroblasts in the remote remodeling myocardium increases. Therefore, study of inflammatory and reparative cell infiltration and assessment of ECM protein deposition should include systematic assessment of each end point in the infarcted region, the peri-infarct area, and the remote remodeling myocardium.

Sympathetic nerves are damaged by permanent coronary occlusion but can regenerate after injury (220). In the setting of chronic MI, regional hyperinnervation around the infarcted region has been observed, and activation of cardiac sympathetic nerves is important in triggering ventricular arrhythmias, and such proarrhythmic action is dependent on the extent of infarction (1, 70, 315, 326). In contrast, after I/R, chondroitin sulfate proteoglycans prevent reinnervation (99, 100). Thus, the model selected for sympathetic nerve evaluation should be taken into consideration and depends on what question is being asked.

MI: intervention considerations.

The effects of interventions on post-MI remodeling can be studied using both nonreperfused MI and reperfused MI/R models (11, 219, 232, 294, 322). Typically, nonreperfused MI yields accentuated dilative remodeling and exacerbated dysfunction compared with a reperfused infarct involving the same vascular territory, reflecting a combination of more extensive infarct and less effective repair. In the reperfused MI/R model, the effects of genetic or pharmacologic interventions implemented early after reperfusion may reflect differences in the extent of acute cardiomyocyte injury rather than differences in wound healing responses. With permanent occlusion MI (assuming a standardized area at risk) or very late reperfusion models, differences in geometry and function of the remodeling heart are independent of acute cardiomyocyte injury and reflect effects on inflammatory, reparative, or fibrotic cascades. In the presence of an occluded coronary artery, the delivery of systemically administered pharmacologic agents to the infarcted region of large animal models may be dependent on formation of collaterals.

While the development of genetically targeted animals (mice, rats, and rabbits) resulted in an explosion of studies dissecting cell biological mechanisms and molecular pathways, large animal models are considered closer to the clinical situation for translational studies to test safety and effectiveness. Optimal study of molecular, cellular, and LV functional end points and interpretation of the findings require understanding of the underlying pathophysiology. Assessment of infarct size is typically the primary end point for investigations examining the mechanisms of cardioprotection. Assessment of chamber dimensions using echocardiography or MRI is crucial to study the progression of adverse remodeling. Systolic and diastolic cardiac geometry and function can be assessed noninvasively using echocardiography (including Doppler ultrasound and speckle tracking), MRI, and hemodynamic assessment. Mechanistic dissection of specific pathways may require inclusion of additional cell physiology and molecular or proteomic end points. In experimental models of MI, understanding the time course of the cellular and molecular events is critical for optimal study design. The effects of varying ischemic intervals on survival and activation of noncardiomyocyte cellular and acellular (e.g., ECM) compartments are poorly understood. Longer coronary occlusion times have distinct effects on cardiac repair, by extending infarct size and by influencing susceptible noncardiomyocyte populations, such as endothelial cells, fibroblasts, pericytes, and immune cells (85). Most studies characterizing responses to myocardial injury have so far been performed in healthy young animals. Comorbid conditions, such as aging, diabetes, and metabolic dysfunction, affect the pattern of ischemic injury and modify the time course and qualitative characteristics of the inflammatory and reparative responses (27, 106, 202, 238, 298, 319, 323). These comorbidities are relevant in the clinical context and must be considered in translation of experimental findings to the clinic.

To summarize, the major strengths and limitations of the nonreperfused and reperfused MI models are shown in Table 1.

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