Florian Bönner

Selective Activation of Adenosine A2A Receptors on Immune Cells by a CD73-Dependent Prodrug Suppresses Joint Inflammation in Experimental Rheumatoid Arthritis

Phosphorylated adenosine A2A receptor agonists suppressed inflammation in a model of arthritis without A2A-mediated vasodilatory side effects. Separating the Wheat from the Chaff Extolling the virtues of simple building design, the modern architect Ludwig Mies van der Rohe famously declared that “less is more,” a philosophy that applies to modern drug design as well. Because simpler drugs have fewer side effects, the promise of adenosine A2A receptor agonists as therapeutics would grow if one could only separate their anti-inflammatory and vasodilator functions. Now, Flögel et al. built an adenosine A2A receptor agonist (chet-AMP) that displays only the anti-inflammatory function in an animal model of rheumatoid arthritis. How the authors accomplished this feat lies “in the details,” to again paraphrase van der Rohe. To isolate the anti-inflammatory effects of A2AR, Flögel et al. synthesized a prodrug that required, for its activation, the presence of ecto-5′-nucleotidase (CD73), which is mainly found on endothelial and immune cells. Using 19F magnetic resonance imaging to track inflammation noninvasively over time, the authors showed that chet-AMP, but not chet-adenosine, reduced inflammation in a mouse model of collagen-induced arthritis. This effect was dependent on the presence of both CD73 and A2AR, and no vasodilation was observed until drug concentrations were increased 100-fold. Physicians most often use corticosteroids to treat inflammatory conditions, but these drugs, although effective, can cause serious complications. By simplifying delivery of a drug only to the sites where it is needed most, Flögel et al. quell inflammation and avoid an undesirable side effect. Adenosine A2A receptor (A2AR) agonists are both highly effective anti-inflammatory agents and potent vasodilators. To separate these two activities, we have synthesized phosphorylated A2AR agonists (prodrugs) that require the presence of ecto-5′-nucleotidase (CD73) to become activated. In the model of collagen-induced arthritis, 2-(cyclohexylethylthio)adenosine 5′-monophosphate (chet-AMP), but not 2-(cyclohexylethylthio)adenosine (chet-adenosine), potently reduced inflammation as assessed by fluorine-19 (19F) magnetic resonance imaging and by histology. The prodrug effect was blunted by inhibition of CD73 and A2AR. The selectivity of drug action is due to profound up-regulation of CD73 and adenosine A2AR expression in neutrophils and inflammatory monocytes as found in recovered cells from the synovial fluid of arthritic mice. Plasma chet-adenosine was in the subnanomolar range when chet-AMP was applied, whereas concentrations required for vasodilation were about 100 times higher. Thus, chet-AMP is a potent immunosuppressant with negligible vasodilatory activity. These data suggest that phosphorylated A2AR agonists may serve as a promising new group of drugs for targeted immunotherapy of inflammation.

19F magnetic resonance imaging of endogenous macrophages in inflammation.

In this article, we review the use of (19) F MRI (magnetic resonance imaging) for in vivo tracking of monocytes and macrophages in the course of tissue inflammation. Emulsified perfluorocarbons (PFCs) are preferentially phagocytized by monocytes/macrophages and are readily detected by (19) F MRI. Because of the lack of any (19) F background in the body, observed signals are robust and exhibit an excellent degree of specificity. As a consequence of progressive infiltration of the labeled immunocompetent cells into inflamed areas, foci of inflammation can be localized as hot spots by simultaneous acquisition of morphologically matched proton ((1) H) and fluorine ((19) F) MRI. The identification of inflammation by (19) F MRI--at a time when the inflammatory cascade is initiated--opens the possibility for an early detection and more timely therapeutic intervention. Since signal intensity in the (19) F images reflects the severity of inflammation, this approach is also suitable to monitor the efficacy of pharmaceutical treatment. Because PFCs are biochemically inert and the fluorine nucleus exhibits high magnetic resonance (MR) sensitivity, (19) F MRI may be applicable for clinical inflammation imaging.

Resident Cardiac Immune Cells and Expression of the Ectonucleotidase Enzymes CD39 and CD73 after Ischemic Injury

Background The ectoenzymes CD39 and CD73 are expressed by a broad range of immune cells and promote the extracellular degradation of nucleotides to anti-inflammatory adenosine. This study explored the abundance of CD73 and CD39 on circulating and resident cardiac leukocytes and coronary endothelial cells under control conditions and in response to inflammation following myocardial ischemia and reperfusion (I/R). Methods and Results A method was elaborated to permit FACS analysis of non-myocardial cells (resident leukocytes, coronary endothelium and CD31− CD45− cells) of the unstressed heart. Under control conditions the murine heart contained 2.3×103 resident leukocytes/mg tissue, the most prominent fraction being antigen-presenting mononuclear cells (CD11b+ CD11c+ F4/80+ MHCII+) followed by B-cells, monocytes and T-cells. CD73 was highly expressed on circulating and resident cardiac lymphoid cells with little expression on myeloid cells, while the opposite was true for CD39. Cardiomyocytes and erythrocytes do not measurably express CD39/CD73 and CD39 dominates on coronary endothelium. Three days after I/R, CD73 was significantly upregulated on invading granulocytes (2.8-fold) and T-cells (1.5-fold). Compared with coronary endothelial cells, CD73 associated with leukocytes comprised 2/3 of the total cardiac CD73. Conclusion Our study suggests that extracellular ATP formed during I/R is preferentially degraded by CD39 present on myeloid cells, while the formation of immunosuppressive adenosine is mainly catalysed by CD73 present on granulocytes and lymphoid cells. Upregulated CD73 on granulocytes and T-cells infiltrating the injured heart is consistent with the existence of an autocrine adenosinergic loop which may promote the healing process.

Myocardial T2 mapping reveals age- and sex-related differences in volunteers

BackgroundT2 mapping indicates to be a sensitive method for detection of tissue oedema hidden beyond the detection limits of T2-weighted Cardiovascular Magnetic Resonance (CMR). However, due to variability of baseline T2 values in volunteers, reference values need to be defined. Therefore, the aim of the study was to investigate the effects of age and sex on quantitative T2 mapping with a turbo gradient-spin-echo (GRASE) sequence at 1.5 T. For that reason, we studied sensitivity issues as well as technical and biological effects on GRASE-derived myocardial T2 maps. Furthermore, intra- and interobserver variability were calculated using data from a large volunteer group.MethodsGRASE-derived multiecho images were analysed using dedicated software. After sequence optimization, validation and sensitivity measurements were performed in muscle phantoms ex vivo and in vivo. The optimized parameters were used to analyse CMR images of 74 volunteers of mixed sex and a wide range of age with typical prevalence of hypertension and diabetes. Myocardial T2 values were analysed globally and according to the 17 segment model. Strain-encoded (SENC) imaging was additionally performed to investigate possible effects of myocardial strain on global or segmental T2 values.ResultsEx vivo studies in muscle phantoms showed, that GRASE-derived T2 values were comparable to those acquired by a standard multiecho spinecho sequence but faster by a factor of 6. Besides that, T2 values reflected tissue water content. The in vivo measurements in volunteers revealed intra- and interobserver correlations with R2=0.91 and R2=0.94 as well as a coefficients of variation of 2.4% and 2.2%, respectively. While global T2 time significantly decreased towards the heart basis, female volunteers had significant higher T2 time irrespective of myocardial region. We found no correlation of segmental T2 values with maximal systolic, diastolic strain or heart rate. Interestingly, volunteers´ age was significantly correlated to T2 time while that was not the case for other coincident cardiovascular risk factors.ConclusionGRASE-derived T2 maps are highly reproducible. However, female sex and aging with typical prevalence of hypertension and diabetes were accompanied by increased myocardial T2 values. Thus, sex and age must be considered as influence factors when using GRASE in a diagnostic manner.

Ecto-5′-Nucleotidase on Immune Cells Protects From Adverse Cardiac Remodeling

Rationale: Ecto-5′-nucleotidase (CD73) on immune cells is emerging as a critical pathway and therapeutic target in cardiovascular and autoimmune disorders. Objective: Here, we investigated the role of CD73 in postinfarction inflammation, cardiac repair, and remodeling in mice after reperfused myocardial infarction (50-minute ischemia). Methods and Results: We found that compared with control mice (1) cardiac function in CD73−/− mice more severely declined after infarction (systolic failure with enhanced myocardial edema formation) as determined by MRI and was associated with the persistence of cardiac immune cell subsets, (2) cardiac adenosine release was augmented 7 days after ischemia/reperfusion in control mice but reduced by 90% in CD73 mutants, (3) impaired healing involves M1-driven immune response with increased tumor necrosis factor-&agr; and interleukin-17, as well as decreased transforming growth factor-&bgr; and interleukin-10, and (4) CD73−/− mice displayed infarct expansion accompanied by an immature replacement scar and diffuse ventricular fibrosis. Studies on mice after bone marrow transplantation revealed that CD73 present on immune cells is a major determinant promoting cardiac healing. Conclusions: These results, together with the upregulation of CD73 on immune cells after ischemia/reperfusion, demonstrate the crucial role of purinergic signaling during cardiac healing and provide groundwork for novel anti-inflammatory strategies in treating adverse cardiac remodeling.

CD73 on Immune Cells Protects from Adverse Cardiac Remodeling

Rationale: Ecto-5′-nucleotidase (CD73) on immune cells is emerging as a critical pathway and therapeutic target in cardiovascular and autoimmune disorders. Objective: Here, we investigated the role of CD73 in postinfarction inflammation, cardiac repair, and remodeling in mice after reperfused myocardial infarction (50-minute ischemia). Methods and Results: We found that compared with control mice (1) cardiac function in CD73−/− mice more severely declined after infarction (systolic failure with enhanced myocardial edema formation) as determined by MRI and was associated with the persistence of cardiac immune cell subsets, (2) cardiac adenosine release was augmented 7 days after ischemia/reperfusion in control mice but reduced by 90% in CD73 mutants, (3) impaired healing involves M1-driven immune response with increased tumor necrosis factor-&agr; and interleukin-17, as well as decreased transforming growth factor-&bgr; and interleukin-10, and (4) CD73−/− mice displayed infarct expansion accompanied by an immature replacement scar and diffuse ventricular fibrosis. Studies on mice after bone marrow transplantation revealed that CD73 present on immune cells is a major determinant promoting cardiac healing. Conclusions: These results, together with the upregulation of CD73 on immune cells after ischemia/reperfusion, demonstrate the crucial role of purinergic signaling during cardiac healing and provide groundwork for novel anti-inflammatory strategies in treating adverse cardiac remodeling.

Visualization of immune cell infiltration in experimental viral myocarditis by 19 F MRI in vivo

ObjectiveThis paper introduces a new approach permitting for the first time a specific, non-invasive diagnosis of myocarditis by visualizing the infiltration of immune cells into the myocardium.Materials and methodsThe feasibility of this approach is shown in a murine model of viral myocarditis. Our study uses biochemically inert perfluorocarbons (PFCs) known to be taken up by circulating monocytes/macrophages after intravenous injection.ResultsIn vivo 19F MRI at 9.4 T demonstrated that PFC-loaded immune cells infiltrate into inflamed myocardial areas. Because of the lack of any fluorine background in the body, detected 19F signals of PFCs are highly specific as confirmed ex vivo by flow cytometry and histology.ConclusionSince PFCs are a family of compounds previously used clinically as blood substitutes, the technique described in our paper holds the potential as a new imaging modality for the diagnosis of myocarditis in man.

T2 mapping in different cardiomyopathies: first clinical experience

Background Structural myocardial changes accompany myocardial pathologies such as myocardial ischemia, myocarditis, hypertrophy (HCM) and myocardial remodeling. These changes affect T2 relaxation times which can noninvasively be detected by cardiovascular magnetic resonance imaging (CMR). Since the interpretation of T2 weighted images remains a “risky business” due to subjectivity, the purpose of our study was to evaluate direct T2 value quantification by T2 mapping in different types of cardiomyopathies. Methods T2 maps were calculated from images recorded with a gated multislice GRASE sequence (9 echos, separated by TE = 7 ms, TR = 750 ms, TA = 5 min, Voxel Size: 2x2x10mm, fat saturation). For creation of T2 maps an exponential decay curve was fitted to the intensity progression of each pixel within the images obtained from the multi echo sequence using a dedicated software based on the graphical programming language LabVIEW (National Instruments, Austin, TX). T2 value distribution in 3 short axis slices (apical, mid-ventricular and basal) were evaluated. Mean, median and standard deviation of values were calculated automatically after manual identification of left ventricular myocardium. T2 maps were analysed in patients with low grade (1450ng/l) high sensitive troponin T (hsTnT) elevation (n=10), myocarditis (n=10), HCM (n=10) and in a young (mean age: 25 years; n=5) as well as elderly

Myocardial T2 Mapping Increases Noninvasive Diagnostic Accuracy for Biopsy-Proven Myocarditis

Detection of myocardial inflammation in patients with clinically suspected acute myocarditis (sAMC) is of prognostic importance but remains a challenge in routine clinical practice [(1)][1]. Compared with endomyocardial biopsy (EMB), the diagnostic gold standard, cardiovascular magnetic resonance (

Technical Advance: Monitoring the trafficking of neutrophil granulocytes and monocytes during the course of tissue inflammation by noninvasive 19F MRI

Inflammation results in the recruitment of neutrophils and monocytes, which is crucial for the healing process. In the present study, we used 19F MRI to monitor in vivo the infiltration of neutrophils and monocytes from the onset of inflammation to the resolution and healing phase. Matrigel, with or without LPS, was s.c.‐implanted into C57BL/6 mice. This resulted in a focal inflammation lasting over a period of 20 days, with constantly decreasing LPS levels in doped matrigel plugs. After i.v. administration of 19F containing contrast agent, 19F MRI revealed a zonular 19F signal in the periphery of LPS containing matrigel plugs, which was not observed in control plugs. Analysis of the 19F signal over the observation period demonstrated the strongest 19F signal after 24 h, which decreased to nearly zero after 20 days. The 19F signal was mirrored by the amount of leukocytes in the matrigel, with neutrophils dominating at early time‐points and macrophages at later time‐points. Both populations were shown to take up the 19F contrast agent. In conclusion, 19F MRI, in combination with the matrigel/LPS model, permits the noninvasive analysis of neutrophil and monocyte infiltration over the complete course of inflammation in vivo.