Linking erythropoietin to Treg-dependent allograft survival through myeloid cells.

Erythropoietin (EPO) has multiple nonerythropoietic functions, including immune modulation, but EPO's effects in transplantation remain incompletely understood. We tested the mechanisms linking EPO administration to prolongation of murine heterotopic heart transplantation using WT and conditional EPO receptor-knockout (EPOR-knockout) mice as recipients. In WT controls, peritransplant administration of EPO synergized with CTLA4-Ig to prolong allograft survival (P < 0.001), reduce frequencies of donor-reactive effector CD8+ T cells in the spleen (P < 0.001) and in the graft (P < 0.05), and increase frequencies and total numbers of donor-reactive Tregs (P < 0.01 for each) versus CTLA4-Ig alone. Studies performed in conditional EPOR-knockout recipients showed that each of these differences required EPOR expression in myeloid cells but not in T cells. Analysis of mRNA isolated from spleen monocytes showed that EPO/EPOR ligation upregulated macrophage-expressed, antiinflammatory, regulatory, and pro-efferocytosis genes and downregulated selected proinflammatory genes. Taken together, the data support the conclusion that EPO promotes Treg-dependent murine cardiac allograft survival by crucially altering the phenotype and function of macrophages. Coupled with our previous documentation that EPO promotes Treg expansion in humans, the data support the need for testing the addition of EPO to costimulatory blockade-containing immunosuppression regimens in an effort to prolong human transplant survival.

T cell-derived tumor necrosis factor induces cytotoxicity by activating RIPK1-dependent target cell death.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1's death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.

Robust effect of metabolic syndrome on major metabolic pathways in the myocardium.

Although the high-fat-diet-induced metabolic syndrome (MetS) is a precursor of human cardiac pathology, the myocardial metabolic state in MetS is far from clear. The discrepancies in metabolite handling between human and small animal models and the difficulties inherent in obtaining human tissue complicate the identification of the myocardium-specific metabolic response in patients. Here we use the large animal model of swine that develops the hallmark criteria of human MetS. Our comparative metabolomics together with transcriptomics and computational nonnegative matrix factorization (NMF) interpretation of the data exposes significant decline in metabolites related to the fatty acid oxidation, glycolysis, and pentose phosphate pathway. Behind the reversal lies decreased expression of enzymes that operate in the pathways. We showed that diminished glycogen deposition is a metabolic signature of MetS in the pig myocardium. The depletion of glycogen arises from disbalance in expression of genes that break down and synthesize glycogen. We show robust acetoacetate accumulation and activated expression of key enzymes in ketone body formation, catabolism and transporters, suggesting a shift in fuel utilization in MetS. A contrasting enrichment in O-GlcNAcylated proteins uncovers hexosamine pathway and O-GlcNAcase (OGA) expression involvement in the myocardial response to MetS. Although the hexosamine biosynthetic pathway (HBP) activity and the availability of the UDP-GlcNAc substrate in the MetS myocardium is low, the level of O-GlcNacylated proteins is high as the O-GlcNacase is significantly diminished. Our data support the perception of transcriptionally driven myocardial alterations in expression of standard fatty acids, glucose metabolism, glycogen, and ketone body related enzymes and subsequent paucity of their metabolite products in MetS. This aberrant energy metabolism in the MetS myocardium provide insight into the pathogenesis of CVD in MetS.

Extracellular Vesicle Injection Improves Myocardial Function and Increases Angiogenesis in a Swine Model of Chronic Ischemia.

BACKGROUND: Mesenchymal stem cell-derived extracellular vesicles (EVs) are believed to be cardioprotective in myocardial infarct. The objective of this study was to examine the effects of human mesenchymal cell-derived EV injection on cardiac function, myocardial blood flow, and vessel density in the setting of chronic myocardial ischemia. METHODS AND RESULTS: Twenty-three Yorkshire swine underwent placement of an ameroid constrictor on their left circumflex artery. Two weeks later, the animals were split into 2 groups: the control group (CON; n=7) and the EV myocardial injection group (MVM; n=10). The MVM group underwent myocardial injection of 50 µg of EVs in 2 mL 0.9% saline into the ischemic myocardium. Five weeks later, the pigs underwent a harvest procedure, and the left ventricular myocardium was analyzed. Absolute blood flow and the ischemic/nonischemic myocardial perfusion ratio were increased in the ischemic myocardium in the MVM group compared with the CON group. Pigs in the MVM group had increased capillary and arteriolar density in the ischemic myocardial tissue compared with CON pigs. There was an increase in expression of the phospho-mitogen-activated protein kinase/mitogen-activated protein kinase ratio, the phospho-endothelial nitric oxide synthase/endothelial nitric oxide synthase ratio, and total protein kinase B in the MVM group compared with CON. There was an increase in cardiac output and stroke volume in the MVM group compared with CON. CONCLUSIONS: In the setting of chronic myocardial ischemia, myocardial injection of human mesenchymal cell-derived EVs increases blood flow to ischemic myocardial tissue by induction of capillary and arteriolar growth via activation of the protein kinase B/endothelial nitric oxide synthase and mitogen-activated protein kinase signaling pathways resulting in increased cardiac output and stroke volume.

Human mannose-binding lectin inhibitor prevents Shiga toxin-induced renal injury.

Hemolytic uremic syndrome caused by Shiga toxin-producing Escherichia coli (STEC HUS) is a worldwide endemic problem, and its pathophysiology is not fully elucidated. Here we tested whether the mannose-binding lectin (MBL2), an initiating factor of lectin complement pathway activation, plays a crucial role in STEC HUS. Using novel human MBL2-expressing mice (MBL2 KI) that lack murine Mbls (MBL2(+/+)Mbl1(-/-)Mbl2(-/-)), a novel STEC HUS model consisted of an intraperitoneal injection with Shiga toxin-2 (Stx-2) with or without anti-MBL2 antibody (3F8, intraperitoneal). Stx-2 induced weight loss, anemia, and thrombocytopenia and increased serum creatinine, free serum hemoglobin, and cystatin C levels, but a significantly decreased glomerular filtration rate compared with control/sham mice. Immunohistochemical staining revealed renal C3d deposition and fibrin deposition in glomeruli in Stx-2-injected mice. Treatment with 3F8 completely inhibited serum MBL2 levels and significantly attenuated Stx-2 induced-renal injury, free serum hemoglobin levels, renal C3d, and fibrin deposition and preserved the glomerular filtration rate. Thus, MBL2 inhibition significantly protected against complement activation and renal injury induced by Stx-2. This novel mouse model can be used to study the role of complement, particularly lectin pathway-mediated complement activation, in Stx-2-induced renal injury.

Human mannose-binding lectin inhibitor prevents myocardial injury and arterial thrombogenesis in a novel animal model.

Myocardial infarction and coagulation disorders are leading causes of disability and death in the world. An important role of the lectin complement pathway in myocardial infarction and coagulation has been demonstrated in mice genetically deficient in lectin complement pathway proteins. However, these studies are limited to comparisons between wild-type and deficient mice and lack the ability to examine reversal/inhibition of injury after disease establishment. We developed a novel mouse that expresses functional human mannose-binding lectin (MBL) 2 under the control of Mbl1 promoter. Serum MBL2 concentrations averaged approximately 3 µg/mL in MBL2(+/+)Mbl1(-/-)Mbl2(-/-) [MBL2 knock in (KI)] mice. Serum MBL2 level in MBL2 KI mice significantly increased after 7 (8 µg/mL) or 14 (9 µg/mL) days of hyperglycemia compared to normoglycemic mice (P < 0.001). Monoclonal antibody 3F8 inhibited C3 deposition on mannan-coated plates in MBL2 KI, but not wild-type, mice. Myocardial ischemia/reperfusion in MBL2 KI mice revealed that 3F8 preserved cardiac function and decreased infarct size and fibrin deposition in a time-dependent manner. Furthermore, 3F8 prevented ferric chloride-induced occlusive arterial thrombogenesis in vivo. MBL2 KI mice represent a novel animal model that can be used to study the lectin complement pathway in acute and chronic models of human disease. Furthermore, these novel mice demonstrate the therapeutic window for MBL2 inhibition for effective treatment of disease and its complications.

Endogenous and natural complement inhibitor attenuates myocardial injury and arterial thrombogenesis.

BACKGROUND: Coagulation disorders and reperfusion of ischemic myocardium are major causes of morbidity and mortality. Lectin pathway initiation complexes are composed of multimolecular carbohydrate recognition subcomponents and 3 lectin pathway-specific serine proteases. We have recently shown that the lectin pathway-specific carbohydrate recognition subcomponent mannose-binding lectin plays an essential role in the pathophysiology of thrombosis and ischemia/reperfusion injury. Thus, we hypothesized that the endogenous mannose-binding lectin (MBL)/ficolin-associated protein-1 (MAP-1) that inhibits complement activation in vitro also could be an in vivo regulator by attenuating myocardial schema/reperfusion injury and thrombogenesis when used at pharmacological doses in wild-type mice. METHODS AND RESULTS: In 2 mouse models, MAP-1 preserves cardiac function, decreases infarct size, decreases C3 deposition, inhibits MBL deposition, and prevents thrombogenesis. Furthermore, we demonstrate that MAP-1 displaces MBL/ficolin-associated serine protease (MASP)-1, MASP-2, and MASP-3 from the MBL complex. CONCLUSIONS: Our results suggest that the natural, endogenous inhibitor MAP-1 effectively inhibits lectin pathway activation in vivo. MAP-1 at pharmacological doses represents a novel therapeutic approach for human diseases involving the lectin pathway and its associated MASPs.

Targeting mannose-binding lectin confers long-lasting protection with a surprisingly wide therapeutic window in cerebral ischemia.

BACKGROUND: The involvement of the complement system in brain injury has been scarcely investigated. Here, we document the pivotal role of mannose-binding lectin (MBL), one of the recognition molecules of the lectin complement pathway, in brain ischemic injury. METHODS AND RESULTS: Focal cerebral ischemia was induced in mice (by permanent or transient middle cerebral artery occlusion) and rats (by 3-vessel occlusion). We first observed that MBL is deposited on ischemic vessels up to 48 hours after injury and that functional MBL/MBL-associated serine protease 2 complexes are increased. Next, we demonstrated that (1) MBL(-/-) mice are protected from both transient and permanent ischemic injury; (2) Polyman2, the newly synthesized mannosylated molecule selected for its binding to MBL, improves neurological deficits and infarct volume when given up to 24 hours after ischemia in mice; (3) anti-MBL-A antibody improves neurological deficits and infarct volume when given up to 18 hours after ischemia, as assessed after 28 days in rats. CONCLUSIONS: Our data show an important role for MBL in the pathogenesis of brain ischemic injury and provide a strong support to the concept that MBL inhibition may be a relevant therapeutic target in humans, one with a wide therapeutic window of application.

Murine hyperglycemic vasculopathy and cardiomyopathy: whole-genome gene expression analysis predicts cellular targets and regulatory networks influenced by mannose binding lectin.

Hyperglycemia, in the absence of type 1 or 2 diabetes, is an independent risk factor for cardiovascular disease. We have previously demonstrated a central role for mannose binding lectin (MBL)-mediated cardiac dysfunction in acute hyperglycemic mice. In this study, we applied whole-genome microarray data analysis to investigate MBL's role in systematic gene expression changes. The data predict possible intracellular events taking place in multiple cellular compartments such as enhanced insulin signaling pathway sensitivity, promoted mitochondrial respiratory function, improved cellular energy expenditure and protein quality control, improved cytoskeleton structure, and facilitated intracellular trafficking, all of which may contribute to the organismal health of MBL null mice against acute hyperglycemia. Our data show a tight association between gene expression profile and tissue function which might be a very useful tool in predicting cellular targets and regulatory networks connected with in vivo observations, providing clues for further mechanistic studies.

Mannose-binding lectin-associated serine protease-1 is a significant contributor to coagulation in a murine model of occlusive thrombosis.

Bleeding disorders and thrombotic complications constitute a major cause of death and disability worldwide. Although it is known that the complement and coagulation systems interact, no studies have investigated the specific role or mechanisms of lectin-mediated coagulation in vivo. FeCl(3) treatment resulted in intra-arterial occlusive thrombogenesis within 10 min in wild-type (WT) and C2/factor B-null mice. In contrast, mannose-binding lectin (MBL)-null and MBL-associated serine protease (MASP)-1/-3 knockout (KO) mice had significantly decreased FeCl(3)-induced thrombogenesis. Reconstitution with recombinant human (rh) MBL restored FeCl(3)-induced thrombogenesis in MBL-null mice to levels comparable to WT mice, suggesting a significant role of the MBL/MASP complex for in vivo coagulation. Additionally, whole blood aggregation demonstrated increased MBL/MASP complex-dependent platelet aggregation. In vitro, MBL/MASP complexes were captured on mannan-coated plates, and cleavage of a chromogenic thrombin substrate (S2238) was measured. We observed no significant differences in S2238 cleavage between WT, C2/factor B-null, MBL-A(-/-), or MBL-C(-/-) sera; however, MBL-null or MASP-1/-3 KO mouse sera demonstrated significantly decreased S2238 cleavage. rhMBL alone failed to cleave S2238, but cleavage was restored when rMASP-1 was added to either MASP-1/-3 KO sera or rhMBL. Taken together, these findings indicate that MBL/MASP complexes, and specifically MASP-1, play a key role in thrombus formation in vitro and in vivo.

Absence of mannose-binding lectin prevents hyperglycemic cardiovascular complications.

Diabetes, stress, pharmaceuticals, surgery, and physical trauma can lead to hyperglycemic conditions. A consistent relationship has been found between chronic inflammation and the cardiovascular complications of hyperglycemia. We hypothesized that cardiomyopathy and vasculopathy resulting from acute hyperglycemia are dependent on mannose-binding lectin (MBL) and lectin complement pathway activation. Hyperglycemia was induced in wild-type (WT) C57BL/6 and MBL-null mice after streptozotocin administration. Echocardiographic data and tissue samples were collected after 4, 7, or 14 days of acute hyperglycemia. Hyperglycemic WT mice demonstrated dilated cardiomyopathy with significantly increased short and long axis area measurements during systole and diastole compared to hyperglycemic MBL-null mice. The EC(50) for acetylcholine-induced relaxation of mesenteric arterioles in WT mice after 4 days of hyperglycemia demonstrated a significant loss of nitric oxide-mediated relaxation compared to normoglycemic WT or hyperglycemic MBL-null mice. Myocardial histochemistry and Western blot analysis revealed a significant influx of macrophages, altered morphology, and increased elastin and collagen deposition in hyperglycemic WT hearts compared to MBL-null hearts. Serum transforming growth factor-ß1 levels were significantly lower in hyperglycemic MBL-null compared to WT mice, suggesting decreased profibrotic signaling. Together, these data suggest that MBL and the lectin complement pathway play a significant role in vascular dysfunction and cardiomyopathy after acute hyperglycemia.

Complement 3 is involved with ventilator-induced lung injury.

Humoral molecules can trigger injury on mechanically stressed and damaged tissue. We have studied the role of complement 3 (C3) in a mouse model of ventilator-induced lung injury (VILI). Compared with sham-treated wild type (WT) mice, ventilated WT mice have reduced total bronchoalveolar lavage (BAL) cells; and elevated activities of thrombin and matrix metalloproteinases (MMPs), such as gelatinase/collagenase in the BAL fluid. In contrast, these parameters in ventilated C3 null mice are not significantly different from sham-treated WT and C3 null mice. In mechanically ventilated mice, thrombin activity and MMPs are lower in C3 null mice than in WT mice and are inversely correlated with total single BAL cells. C3 activation is associated with MMP activation in vitro. Pretreatment of WT mice with humanized cobra venom factor, which inactivates C3, reduces C3 deposition in the lung and increases total BAL cells in VILI. We propose that C3 is involved with VILI and inhibition of complement activation may be a potential therapeutic strategy.