Anti-inflammatory Effects of Extracellular Cyclosporins Are Exclusively Mediated by CD147

Leukocyte trafficking and recruitment is a critical process in host immune surveillance and in inflammatory diseases. Extracellular cyclophilins (eCyps) have been identified as a novel class of chemotactic mediators. The impact of eCyp/CD147 interactions for the recruitment of leukocytes during inflammation was analyzed using a structurally simplified cell-impermeable eCyp inhibitor. This compound was highly effective at inhibiting leukocyte migration toward CypA in vitro as well as in the recruitment of leukocytes during inflammation in a mouse model of experimentally induced peritonitis and delayed-type hypersensitivity reaction. By using CD147–/– mice in combination with the cell-impermeable eCyp inhibitor, we were able to show that the action of eCyps in inflammation is exclusively mediated by interaction with CD147. Our findings suggest that blocking eCyps may be an effective therapeutic target for reducing inflammatory diseases associated with leukocyte recruitment

MM284 reduces infiltration of T-cells and macrophages in autoimmune myocarditis in mice.

<p><b>A</b>, <b>B</b>, Infiltration of T-cells and macrophages was assessed using anti-CD3 and anti-Mac-3 staining. Representative images for each treatment are shown. Data in the right panels show individual scoring results (n ≥ 10), horizontal bars indicate medians, * indicates p < 0.05.</p

Extracellular Cyclophilin A—inhibition by MM284 attenuates migration of human monocytes <i>in vitro</i>.

<p><b>A</b>, Effects of MM284 on Cyclophilin A-induced chemotaxis were studied using a modified Boyden chamber. Migration of human monocytes was assessed using media, SDF-1α (50ng/ml) as positive control, CyPA (200nM), or CyPA (200nM) + MM284 (200; 500; and 800nM). Cells were incubated for 4h at 37°C. Migrated cells were counted and a chemotactic index was calculated (n = 5). <b>B</b>, Adhesion of monocytes under flow conditions to activated human umbilical vein endothelial cells. Data are shown as mean ± SEM. <b>C</b>, Cell membrane permeability of MM284 was assessed using a competition assay. MM284 or NIM811 (a cell permeable CsA derivate) was used to displace Fluo-mCsA (a fluorescently labeled (green), cell permeable CsA derivate) from the cytoplasm of THP1 cells. The presence of Fluo-mCsA in the cytoplasm after treatment was assessed using confocal laser scanning microscopy. * indicates p < 0.05 compared to CyPA 200nM.</p

MM284 reduces cardiac inflammation and fibrosis in autoimmune myocarditis in mice.

<p>Troponin I-induced autoimmune myocarditis was studied in A/J mice. <b>A</b>, Hematoxylin and Eosin staining (H&E) was used to determine the area of cardiac damage 28 days after induction of myocarditis. <b>B</b>, Masson’s Trichrome staining was used to determine the extent of fibrotic remodeling in the myocardium 28 days after induction of experimental autoimmune myocarditis. Representative images for each treatment are shown. Data in the right panels show individual scoring results (n ≥ 10), horizontal bars indicate medians, * indicates p < 0.05. <b>C</b>, 28 days after induction of autoimmune myocarditis, small animal echocardiography was used to evaluate left ventricular function (n = 7). Left ventricular ejection fraction (EF), fractional shortening (FS), left ventricular mass (LV Mass), left ventricular end-systolic volume (LV ESV), left ventricular end-diastolic volume (LV EDV), left ventricular posterior wall in diastole (LVPWd), heart rate, and body weight are shown. n.s. indicates not significant.</p

Influence of MM284 on myocardial TNFα, IL-6 and MMP-9 expression.

<p>Realtime PCR assay was performed to evaluate expression of TNFα (<b>A</b>), IL-6 (<b>B</b>) and MMP-9 (<b>C</b>) in the myocardium. Data are shown as mean of relative expression ± SD, n ≥ 8. * indicates p < 0.05, n.s. indicates not significant.</p

Extracellular Cyclophilin A is associated with myocardial fibrosis.

<p><b>A</b>, Representative stainings of heart sections from mice 28 days after induction of troponin I-induced autoimmune myocarditis. Sections were stained with Masson’s Trichrome, anti-CyPA, and IgG-control as indicated. Marked area is magnified in the middle panel. <b>B</b>, Representative image of healthy control mice stained with anti-CyPA or IgG control. <b>C</b>, Myocardial stainings from mice 28 days after induction of troponin I-induced autoimmune myocarditis. Myocardial sections were stained with anti-CyPA (green), rhodamin phalloidin (red) for actin cytoskeleton, and ToPro-3 for nuclei (blue), as described in materials and methods. Arrows indicate localization of CyPA in the extracellular space.</p