Johannes Weirather

Activation of CD4+ T Lymphocytes Improves Wound Healing and Survival After Experimental Myocardial Infarction in Mice

Background— The role of adaptive immunity, especially CD4+ T-helper cells, has not yet been systematically investigated in wound healing and remodeling after myocardial infarction (MI). Therefore, we studied whether CD4+ T cells become activated and influence wound healing after experimental MI in mice. Methods and Results— When we compared sham versus MI in wild-type (WT) mice, T-cell receptor–dependent activation of both conventional Foxp3− and regulatory Foxp3+ CD4+ T cells could be demonstrated in heart-draining lymph nodes within the first week after MI. Concomitantly, we found infiltration of CD4+ T cells in infarcted myocardium. To study the role of CD4+ T cells in wound healing and remodeling, CD4+ T-cell–deficient mice (CD4 knockout [KO], MHCII&Dgr;/&Dgr;) and T-cell receptor–transgenic OT-II mice recognizing an irrelevant ovalbumin-derived peptide were studied. Serial echocardiography up to day 56 after MI revealed increased left ventricular dilation in CD4 KO compared with WT mice. Within the infarcted myocardium, CD4 KO mice displayed higher total numbers of leukocytes and proinflammatory monocytes (18.3±3.0 104/mg WT versus 75.7±17.0 104/mg CD4 KO, P<0.05). MHCII&Dgr;/&Dgr; and OT-II mice displayed significantly greater mortality (21% WT versus 48% OT-II, P<0.05, and WT 22% versus 52% MHCII&Dgr;/&Dgr;, P<0.05) and myocardial rupture rates than WT mice. Collagen matrix formation in the infarct zone was severely disturbed in CD4 KO and MHCII&Dgr;/&Dgr; mice, as well as in OT-II mice. Conclusions— The present study provides the first evidence that CD4+ T cells become activated after MI, presumably driven by recognition of cardiac autoantigens, and facilitate wound healing of the myocardium.

Foxp3+ CD4+ T Cells Improve Healing After Myocardial Infarction by Modulating Monocyte/Macrophage Differentiation

Rationale: An exaggerated or persistent inflammatory activation after myocardial infarction (MI) leads to maladaptive healing and subsequent remodeling of the left ventricle. Foxp3+ CD4+ regulatory T cells (Treg cells) contribute to inflammation resolution. Therefore, Treg cells might influence cardiac healing post-MI. Objective: Our aim was to study the functional role of Treg cells in wound healing post-MI in a mouse model of permanent left coronary artery ligation. Methods and Results: Using a model of genetic Treg-cell ablation (Foxp3DTR mice), we depleted the Treg-cell compartment before MI induction, resulting in aggravated cardiac inflammation and deteriorated clinical outcome. Mechanistically, Treg-cell depletion was associated with M1-like macrophage polarization, characterized by decreased expression of inflammation-resolving and healing-promoting factors. The phenotype of exacerbated cardiac inflammation and outcome in Treg-cell–ablated mice could be confirmed in a mouse model of anti-CD25 monoclonal antibody–mediated depletion. In contrast, therapeutic Treg-cell activation by superagonistic anti-CD28 monoclonal antibody administration 2 days after MI led to improved healing and survival. Compared with control animals, CD28-SA–treated mice showed increased collagen de novo expression within the scar, correlating with decreased rates of left ventricular ruptures. Therapeutic Treg-cell activation induced an M2-like macrophage differentiation within the healing myocardium, associated with myofibroblast activation and increased expression of monocyte/macrophage-derived proteins fostering wound healing. Conclusions: Our data indicate that Treg cells beneficially influence wound healing after MI by modulating monocyte/macrophage differentiation. Moreover, therapeutic activation of Treg cells constitutes a novel approach to improve healing post-MI.

Specific somatostatin receptor II expression in arterial plaque: 68Ga-DOTATATE autoradiographic, immunohistochemical and flow cytometric studies in apoE-deficient mice

BACKGROUND The rupture of atherosclerotic plaques is triggered by inflammation. Specific detection of inflammation is therefore the focus of many investigations. Noninvasive imaging methods, such as positron emission tomography (PET), also are suited for this purpose. (68)Ga-DOTATATE is a (68)Ga-labeled radiotracer with specific affinity to somatostatin receptor subtype-2 (SSTR-2). SSTR-2 was found specifically expressed on human macrophages/monocytes. OBJECTIVE We aimed to confirm the distribution of SSTR-2 in inflammatory plaques, and to assess its co-localization with macrophages within the plaques. We also assessed (68)Ga-DOTATATE uptakes in plaques by autoradiography. METHOD Apolipoprotein E (ApoE)-/- mice on a high-cholesterol diet were injected with (68)Ga-DOTATATE. The animals were sacrificed and aorta sections were examined using autoradiography and immunohistochemistry. Furthermore, expression of SSTR-2 was analyzed by flow cytometry. Western blot was conducted to assess SSTR-2 regulation in basal and lipopolysaccharide (LPS)-activated state. To evaluate the specificity of the (68)Ga-DOTATATE, the sections were pre-incubated with monoclonal SSTR-2 antibody before autoradiography. RESULT Autoradiographic imaging showed uptake of (68)Ga-DOTATATE co-localized with the macrophage-rich plaques by immunohistochemical examination. A high expression of SSTR-2 on macrophages was found by flow cytometry and western blot. Stimulation with lipopolysaccharide did not alter expression of SSTR-2 in macrophages. CONCLUSION Due to its specific binding to macrophages, (68)Ga-DOTATATE might be a suitable radiotracer for the evaluation of inflammatory activity in unstable plaques.

Targeting P-Selectin by Gallium-68–Labeled Fucoidan Positron Emission Tomography for Noninvasive Characterization of Vulnerable Plaques Correlation With In Vivo 17.6T MRI

Objective— Nuclear imaging of active plaques still remains challenging. Advanced atherosclerotic plaques have a strong expression of P-selectin by the endothelium overlying active atherosclerotic plaques, but not on the endothelium overlying inactive fibrous plaques. We proposed a new approach for noninvasive in vivo characterization of P-selectin on active plaques based on 68Ga-Fucoidan, which is a polysaccharidic ligand of P-selectin with a nanomolar affinity. Approach and Results— 68Ga-Fucoidan was tested for its potential to discriminate vulnerable plaques on apolipoprotein E–deficient mice receiving a high cholesterol diet by positron emission tomography and in correlation with 17.6T MRI. Furthermore, 68Ga-Fucoidan was evaluated on endothelial cells in vitro and ex vivo on active plaques using autoradiography. The cellular uptake rate was increased ≈2-fold by lipopolysaccharide induction. Interestingly, on autoradiography, more intensive tracer accumulation at active plaques with thin fibrous caps and high-density foam cells were observed in comparison with a weaker focal uptake in inactive fibrous plaque segments (R=1.7±0.3; P<0.05) and fatty streaks (R=2.4±0.4; P<0.01). Strong uptake of radiotracer colocalized with increased P-selectin expression and high-density macrophage. Focal vascular uptake (mean of target to background ratio=5.1±0.8) of 68Ga-Fucoidan was detected in all apolipoprotein E–deficient mice. Anatomic structures of plaque were confirmed by 17.6T MRI. The autoradiography showed a good agreement of 68Ga-Fucoidan uptake with positron emission tomography. Conclusions— Our data suggest that 68Ga-Fucoidan represents a versatile imaging biomarker for P-selectin with the potential to specifically detect P-selectin expression using positron emission tomography and to discriminate vulnerable plaques in vivo.

Interleukin-13 Deficiency Aggravates Healing and Remodeling in Male Mice After Experimental Myocardial Infarction

Background—Activation of innate immunity, especially infiltration of monocytes, is critical for proper wound healing and scar formation after myocardial infarction (MI). Therefore, we tested the hypothesis that interleukin-13 (IL-13), which influences the differentiation of monocytes/macrophages and has profibrotic properties, modulates wound healing and remodeling after MI. Methods and Results—MI was induced by permanent ligation of the left coronary artery in both male and female wild-type (WT)/IL-13−/− mice. Real-time polymerase chain reaction demonstrated that expression of IL-13 was induced in left and right ventricular myocardium of WT mice within days in response to MI. Fifty-six–day survival was significantly impaired (65% in WT versus 34% in IL-13−/−) in male but not female IL-13−/− (55% in WT versus 54% in IL-13−/−) mice. Serial echocardiography showed significantly increased left ventricular dilation in male IL-13−/− compared with WT mice starting from day 1 after MI, despite comparable infarct size. Fluorescence-activated cell sorter analysis revealed less leukocyte infiltration in male IL-13−/− mice on day 3. Real-time polymerase chain reaction analysis demonstrated reduced expression of marker genes of alternative activation in monocytes sorted from the infarct zone of male IL-13−/− in comparison with WT mice on day 3 after MI. Conclusions—Genetic deficiency of IL-13 worsens outcome after MI in male mice. Our data indicate that IL-13 regulates leukocyte recruitment and induces M2-like monocyte/macrophage differentiation, which modifies wound healing within the infarct zone.

Myocardial aging as a T-cell–mediated phenomenon

Significance Aging is a risk factor for heart diseases, and it is also known to impact on several immunological processes. Nevertheless, most studies addressing the cardio-immune cross-talk have focused on juvenile rather than senescent animal models. In the present study, we addressed this gap and found that immunological activity contributes to myocardial aging. By using different lymphocyte-deficient animal models and heterochronic adoptive cell-transfer protocols, our study revealed a pivotal role for CD4+ T cells in mediating spontaneous local inflammation and mild organ dysfunction in aged hearts. These results might shed new light on the emerging field of “immunocardiology” because they reveal that spontaneous heart-directed immune responses arise even in the absence of previous myocardial tissue damage. In recent years, the myocardium has been rediscovered under the lenses of immunology, and lymphocytes have been implicated in the pathogenesis of cardiomyopathies with different etiologies. Aging is an important risk factor for heart diseases, and it also has impact on the immune system. Thus, we sought to determine whether immunological activity would influence myocardial structure and function in elderly mice. Morphological, functional, and molecular analyses revealed that the age-related myocardial impairment occurs in parallel with shifts in the composition of tissue-resident leukocytes and with an accumulation of activated CD4+ Foxp3− (forkhead box P3) IFN-γ+ T cells in the heart-draining lymph nodes. A comprehensive characterization of different aged immune-deficient mouse strains revealed that T cells significantly contribute to age-related myocardial inflammation and functional decline. Upon adoptive cell transfer, the T cells isolated from the mediastinal lymph node (med-LN) of aged animals exhibited increased cardiotropism, compared with cells purified from young donors or from other irrelevant sites. Nevertheless, these cells caused rather mild effects on cardiac functionality, indicating that myocardial aging might stem from a combination of intrinsic and extrinsic (immunological) factors. Taken together, the data herein presented indicate that heart-directed immune responses may spontaneously arise in the elderly, even in the absence of a clear tissue damage or concomitant infection. These observations might shed new light on the emerging role of T cells in myocardial diseases, which primarily affect the elderly population.

Endothelial Actions of ANP Enhance Myocardial Inflammatory Infiltration in the Early Phase After Acute Infarction

RATIONALE In patients after acute myocardial infarction (AMI), the initial extent of necrosis and inflammation determine clinical outcome. One early event in AMI is the increased cardiac expression of atrial natriuretic peptide (NP) and B-type NP, with their plasma levels correlating with severity of ischemia. It was shown that NPs, via their cGMP-forming guanylyl cyclase-A (GC-A) receptor and cGMP-dependent kinase I (cGKI), strengthen systemic endothelial barrier properties in acute inflammation. OBJECTIVE We studied whether endothelial actions of local NPs modulate myocardial injury and early inflammation after AMI. METHODS AND RESULTS Necrosis and inflammation after experimental AMI were compared between control mice and littermates with endothelial-restricted inactivation of GC-A (knockout mice with endothelial GC-A deletion) or cGKI (knockout mice with endothelial cGKI deletion). Unexpectedly, myocardial infarct size and neutrophil infiltration/activity 2 days after AMI were attenuated in knockout mice with endothelial GC-A deletion and unaltered in knockout mice with endothelial cGKI deletion. Molecular studies revealed that hypoxia and tumor necrosis factor-α, conditions accompanying AMI, reduce the endothelial expression of cGKI and enhance cGMP-stimulated phosphodiesterase 2A (PDE2A) levels. Real-time cAMP measurements in endothelial microdomains using a novel fluorescence resonance energy transfer biosensor revealed that PDE2 mediates NP/cGMP-driven decreases of submembrane cAMP levels. Finally, intravital microscopy studies of the mouse cremaster microcirculation showed that tumor necrosis factor-α-induced endothelial NP/GC-A/cGMP/PDE2 signaling impairs endothelial barrier functions. CONCLUSIONS Hypoxia and cytokines, such as tumor necrosis factor-α, modify the endothelial postreceptor signaling pathways of NPs, with downregulation of cGKI, induction of PDE2A, and altered cGMP/cAMP cross talk. Increased expression of PDE2 can mediate hyperpermeability effects of paracrine endothelial NP/GC-A/cGMP signaling and facilitate neutrophil extravasation during the early phase after MI.

CD4+ Foxp3+ T-cells contribute to myocardial ischemia-reperfusion injury

OBJECTIVE The present study analyzed the effect of CD4+ Forkhead box protein 3 negative (Foxp3-) T-cells and Foxp3+ CD4+ T-cells on infarct size in a mouse myocardial ischemia-reperfusion model. APPROACH AND RESULTS We examined the infarct size as a fraction of the area-at-risk as primary study endpoint in mice after 30minutes of coronary ligation followed by 24hours of reperfusion. CD4+ T-cell deficient MHC-II KO mice showed smaller histologically determined infarct size (34.5±4.7% in MHCII KO versus 59.4±4.9% in wildtype (WT)) and better preserved ejection fraction determined by magnetic resonance tomography (56.9±2.8% in MHC II KO versus 39.0±4.2% in WT). MHC-II KO mice also displayed better microvascular perfusion than WT mice after 24hours of reperfusion. Also CD4+ T-cell sufficient OT-II mice, which express an in this context irrelevant T-cell receptor, revealed smaller infarct sizes compared to WT mice. However, MHC-II blocking anti-I-A/I-E antibody treatment was not able to reduce infarct size indicating that autoantigen recognition is not required for the activation of CD4+ T-cells during reperfusion. Flow-cytometric analysis also did not detect CD4+ T-cell activation in heart draining lymph nodes in response to 24hours of ischemia-reperfusion. Adoptive transfer of CD4+ T-cells in CD4 KO mice increased the infarct size only when including the Foxp3+ CD25+ subset. Depletion of CD4+ Foxp3+ T-cells in DEREG mice enabling specific conditional ablation of this subset by treatment with diphtheria toxin attenuated infarct size as compared to diphtheria toxin treated WT mice. CONCLUSIONS CD4+ Foxp3+ T-cells enhance myocardial ischemia-reperfusion injury. CD4+ T-cells exert injurious effects without the need for prior activation by MHC-II restricted autoantigen recognition.

Role of the Innate Immune System in Ischemic Heart Failure

Abstract Cardiac remodeling after myocardial infarction (MI) is characterized by deleterious changes in cardiac geometry and constitutes the prevailing pathomechanism in the development of heart failure. Infarct size and the quality of infarct healing represent major remodeling determinants and dictate the severity of maladaptations. After ischemic injury, both coagulative necrosis and a high reactive oxygen burden trigger the immune response comprising humoral and cellular effectors that pivotally modulate postinfarction healing. Notably, a fine-balance between the inflammatory response and inflammation resolution is vital to safeguard proper tissue replacement while preventing an adverse exuberant remodeling. The concept that immunity modulates healing and remodeling after MI has been corroborated over the last decades. Numerous experimental studies indicate that successful immunomodulation improves the clinical outcome post-MI. Where therapeutic approaches have been widely disappointing in the clinical arena, manipulating the immune system may yet constitute a promising modality to ameliorate remodeling in patients with MI.