NKG2D receptor activation drives primary graft dysfunction severity and poor lung transplantation outcomes.

Clinical outcomes after lung transplantation, a life-saving therapy for patients with end-stage lung diseases, are limited by primary graft dysfunction (PGD). PGD is an early form of acute lung injury with no specific pharmacologic therapies. Here, we present a large multicenter study of plasma and bronchoalveolar lavage (BAL) samples collected on the first posttransplant day, a critical time for investigations of immune pathways related to PGD. We demonstrated that ligands for NKG2D receptors were increased in the BAL from participants who developed severe PGD and were associated with increased time to extubation, prolonged intensive care unit length of stay, and poor peak lung function. Neutrophil extracellular traps (NETs) were increased in PGD and correlated with BAL TNF-α and IFN-γ cytokines. Mechanistically, we found that airway epithelial cell NKG2D ligands were increased following hypoxic challenge. NK cell killing of hypoxic airway epithelial cells was abrogated with NKG2D receptor blockade, and TNF-α and IFN-γ provoked neutrophils to release NETs in culture. These data support an aberrant NK cell/neutrophil axis in human PGD pathogenesis. Early measurement of stress ligands and blockade of the NKG2D receptor hold promise for risk stratification and management of PGD.

Hypoimmune induced pluripotent stem cell-derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice.

The emerging field of regenerative cell therapy is still limited by the few cell types that can reliably be differentiated from pluripotent stem cells and by the immune hurdle of commercially scalable allogeneic cell therapeutics. Here, we show that gene-edited, immune-evasive cell grafts can survive and successfully treat diseases in immunocompetent, fully allogeneic recipients. Transplanted endothelial cells improved perfusion and increased the likelihood of limb preservation in mice with critical limb ischemia. Endothelial cell grafts transduced to express a transgene for alpha1-antitrypsin (A1AT) successfully restored physiologic A1AT serum levels in mice with genetic A1AT deficiency. This cell therapy prevented both structural and functional changes of emphysematous lung disease. A mixture of endothelial cells and cardiomyocytes was injected into infarcted mouse hearts, and both cell types orthotopically engrafted in the ischemic areas. Cell therapy led to an improvement in invasive hemodynamic heart failure parameters. Our study supports the development of hypoimmune, universal regenerative cell therapeutics for cost-effective treatments of major diseases.

Natural killer cells activated through NKG2D mediate lung ischemia-reperfusion injury.

Pulmonary ischemia-reperfusion injury (IRI) is a clinical syndrome of acute lung injury that occurs after lung transplantation or remote organ ischemia. IRI causes early mortality and has no effective therapies. While NK cells are innate lymphocytes capable of recognizing injured cells, their roles in acute lung injury are incompletely understood. Here, we demonstrated that NK cells were increased in frequency and cytotoxicity in 2 different IRI mouse models. We showed that NK cells trafficked to the lung tissue from peripheral reservoirs and were more mature within lung tissue. Acute lung ischemia-reperfusion injury was blunted in a NK cell-deficient mouse strain but restored with adoptive transfer of NK cells. Mechanistically, NK cell NKG2D receptor ligands were induced on lung endothelial and epithelial cells following IRI, and antibody-mediated NK cell depletion or NKG2D stress receptor blockade abrogated acute lung injury. In human lung tissue, NK cells were increased at sites of ischemia-reperfusion injury and activated NK cells were increased in prospectively collected human bronchoalveolar lavage in subjects with severe IRI. These data support a causal role for recipient peripheral NK cells in pulmonary IRI via NK cell NKG2D receptor ligation. Therapies targeting NK cells may hold promise in acute lung injury.

Complement activation on endothelium initiates antibody-mediated acute lung injury.

Antibodies targeting human leukocyte antigen (HLA)/major histocompatibility complex (MHC) proteins limit successful transplantation and transfusion, and their presence in blood products can cause lethal transfusion-related acute lung injury (TRALI). It is unclear which cell types are bound by these anti-leukocyte antibodies to initiate an immunologic cascade resulting in lung injury. We therefore conditionally removed MHC class I (MHC I) from likely cellular targets in antibody-mediated lung injury. Only the removal of endothelial MHC I reduced lung injury and mortality, related mechanistically to absent endothelial complement fixation and lung platelet retention. Restoration of endothelial MHC I rendered MHC I-deficient mice susceptible to lung injury. Neutrophil responses, including neutrophil extracellular trap (NET) release, were intact in endothelial MHC I-deficient mice, whereas complement depletion reduced both lung injury and NETs. Human pulmonary endothelial cells showed high HLA class I expression, and posttransfusion complement activation was increased in clinical TRALI. These results indicate that the critical source of antigen for anti-leukocyte antibodies is in fact the endothelium, which reframes our understanding of TRALI as a rapid-onset vasculitis. Inhibition of complement activation may have multiple beneficial effects of reducing endothelial injury, platelet retention, and NET release in conditions where antibodies trigger these pathogenic responses.

Cystic fibrosis transmembrane conductance regulator dysfunction in platelets drives lung hyperinflammation.

Cystic fibrosis (CF) lung disease is characterized by an inflammatory response that can lead to terminal respiratory failure. The cystic fibrosis transmembrane conductance regulator (CFTR) is mutated in CF, and we hypothesized that dysfunctional CFTR in platelets, which are key participants in immune responses, is a central determinant of CF inflammation. We found that deletion of CFTR in platelets produced exaggerated acute lung inflammation and platelet activation after intratracheal LPS or Pseudomonas aeruginosa challenge. CFTR loss of function in mouse or human platelets resulted in agonist-induced hyperactivation and increased calcium entry into platelets. Inhibition of the transient receptor potential cation channel 6 (TRPC6) reduced platelet activation and calcium flux, and reduced lung injury in CF mice after intratracheal LPS or Pseudomonas aeruginosa challenge. CF subjects receiving CFTR modulator therapy showed partial restoration of CFTR function in platelets, which may be a convenient approach to monitoring biological responses to CFTR modulators. We conclude that CFTR dysfunction in platelets produces aberrant TRPC6-dependent platelet activation, which is a major driver of CF lung inflammation and impaired bacterial clearance. Platelets and TRPC6 are what we believe to be novel therapeutic targets in the treatment of CF lung disease.

Mitochondrial DNA Stimulates TLR9-Dependent Neutrophil Extracellular Trap Formation in Primary Graft Dysfunction.

The immune system is designed to robustly respond to pathogenic stimuli but to be tolerant to endogenous ligands to not trigger autoimmunity. Here, we studied an endogenous damage-associated molecular pattern, mitochondrial DNA (mtDNA), during primary graft dysfunction (PGD) after lung transplantation. We hypothesized that cell-free mtDNA released during lung ischemia-reperfusion triggers neutrophil extracellular trap (NET) formation via TLR9 signaling. We found that mtDNA increases in the BAL fluid of experimental PGD (prolonged cold ischemia followed by orthotopic lung transplantation) and not in control transplants with minimal warm ischemia. The adoptive transfer of mtDNA into the minimal warm ischemia graft immediately before lung anastomosis induces NET formation and lung injury. TLR9 deficiency in neutrophils prevents mtDNA-induced NETs, and TLR9 deficiency in either the lung donor or recipient decreases NET formation and lung injury in the PGD model. Compared with human lung transplant recipients without PGD, severe PGD was associated with high levels of BAL mtDNA and NETs, with evidence of relative deficiency in DNaseI. We conclude that mtDNA released during lung ischemia-reperfusion triggers TLR9-dependent NET formation and drives lung injury. In PGD, DNaseI therapy has a potential dual benefit of neutralizing a major NET trigger (mtDNA) in addition to dismantling pathogenic NETs.

LPS-induced Lung Platelet Recruitment Occurs Independently from Neutrophils, PSGL-1, and P-Selectin.

Platelets are recruited to inflammatory foci and contribute to host defense and inflammatory responses. Compared with platelet recruitment in hemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defense are poorly understood. Neutrophil recruitment to lung airspaces after inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice after LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils. BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and, 48 hours later, lungs were collected and platelets and neutrophils quantified in tissue sections by immunohistochemistry. The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules, P-selectin or PSGL-1, or treatment with a neutrophil-depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre × mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs. Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation. In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.

Maladaptive role of neutrophil extracellular traps in pathogen-induced lung injury.

Neutrophils dominate the early immune response in pathogen-induced acute lung injury, but efforts to harness their responses have not led to therapeutic advancements. Neutrophil extracellular traps (NETs) have been proposed as an innate defense mechanism responsible for pathogen clearance, but there are concerns that NETs may induce collateral damage to host tissues. Here, we detected NETs in abundance in mouse models of severe bacterial pneumonia/acute lung injury and in human subjects with acute respiratory distress syndrome (ARDS) from pneumonia or sepsis. Decreasing NETs reduced lung injury and improved survival after DNase I treatment or with partial protein arginine deiminase 4 deficiency (PAD4+/-). Complete PAD4 deficiency (PAD4-/-) reduced NETs and lung injury but was counterbalanced by increased bacterial load and inflammation. Importantly, we discovered that the lipoxin pathway could be a potent modulator of NET formation, and that mice deficient in the lipoxin receptor (Fpr2-/-) produced excess NETs leading to increased lung injury and mortality. Lastly, we observed in humans that increased plasma NETs were associated with ARDS severity and mortality, and lower plasma DNase I levels were associated with the development of sepsis-induced ARDS. We conclude that a critical balance of NETs is necessary to prevent lung injury and to maintain microbial control, which has important therapeutic implications.

Mirasol pathogen reduction technology treatment of human whole blood does not induce acute lung injury in mice.

In resource-limited settings and in the military theater, fresh human whole blood is commonly transfused, but infectious risks are a concern. Sophisticated molecular testing for potential infectious agents in the whole blood is often unavailable. To address this unmet need, pathogen reduction technology (PRT) has been developed, and it is an effective approach to inactivate a broad range of pathogens found in human blood. However, studies are needed to determine if it is harmful to blood cells and whether these cells could damage the transfused recipient, including the development of acute lung injury/acute respiratory distress syndrome. In this study, we used a commercial PRT system to treat human whole blood that was then transfused into immunodeficient mice, and the development of acute lung injury was determined. In a model of transfusion-related acute lung injury (TRALI), BALB/c SCID mice developed more robust lung injury when challenged with a MHC Class I monoclonal antibody compared to BALB/c wild-type and NOD/SCID mice. Transfusion of control versus Mirasol PRT-treated whole blood (25% blood volume exchange) into BALB/c SCID mice did not produce lung injury at storage day 1. However, mild lung injury at storage days 14 and 21 was observed without significant differences in lung injury measurements between Mirasol PRT-treated and control groups. The mild storage-dependent acute lung injury correlated with trends for increased levels of cell-free hemoglobin that accumulated in both the control and Mirasol PRT-treated groups. Neutrophil extracellular traps were elevated in the plasma of BALB/c SCID mice in the monoclonal antibody TRALI model, but were not different in mice that received exchange transfusions. In conclusion, exchange transfusion of human whole blood into immunodeficient mice produces mild lung injury that is storage-dependent and not related to pathogen reduction treatment.

The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors.

Platelets are critical for haemostasis, thrombosis, and inflammatory responses, but the events that lead to mature platelet production remain incompletely understood. The bone marrow has been proposed to be a major site of platelet production, although there is indirect evidence that the lungs might also contribute to platelet biogenesis. Here, by directly imaging the lung microcirculation in mice, we show that a large number of megakaryocytes circulate through the lungs, where they dynamically release platelets. Megakaryocytes that release platelets in the lungs originate from extrapulmonary sites such as the bone marrow; we observed large megakaryocytes migrating out of the bone marrow space. The contribution of the lungs to platelet biogenesis is substantial, accounting for approximately 50% of total platelet production or 10 million platelets per hour. Furthermore, we identified populations of mature and immature megakaryocytes along with haematopoietic progenitors in the extravascular spaces of the lungs. Under conditions of thrombocytopenia and relative stem cell deficiency in the bone marrow, these progenitors can migrate out of the lungs, repopulate the bone marrow, completely reconstitute blood platelet counts, and contribute to multiple haematopoietic lineages. These results identify the lungs as a primary site of terminal platelet production and an organ with considerable haematopoietic potential.

Telomere dysfunction in alveolar epithelial cells causes lung remodeling and fibrosis.

Telomeres are short in type II alveolar epithelial cells (AECs) of patients with idiopathic pulmonary fibrosis (IPF). Whether dysfunctional telomeres contribute directly to development of lung fibrosis remains unknown. The objective of this study was to investigate whether telomere dysfunction in type II AECs, mediated by deletion of the telomere shelterin protein TRF1, leads to pulmonary fibrosis in mice (SPC-Cre TRF1fl/fl mice). Deletion of TRF1 in type II AECs for 2 weeks increased γH2AX DNA damage foci, but not histopathologic changes in the lung. Deletion of TRF1 in type II AECs for up to 9 months resulted in short telomeres and lung remodeling characterized by increased numbers of type II AECs, α-smooth muscle actin+ mesenchymal cells, collagen deposition, and accumulation of senescence-associated ß-galactosidase+ lung epithelial cells. Deletion of TRF1 in collagen-expressing cells caused pulmonary edema, but not fibrosis. These results demonstrate that prolonged telomere dysfunction in type II AECs, but not collagen-expressing cells, leads to age-dependent lung remodeling and fibrosis. We conclude that telomere dysfunction in type II AECs is sufficient to cause lung fibrosis, and may be a dominant molecular defect causing IPF. SPC-Cre TRF1fl/fl mice will be useful for assessing cellular and molecular mechanisms of lung fibrosis mediated by telomere dysfunction.

Transfusion of Human Platelets Treated with Mirasol Pathogen Reduction Technology Does Not Induce Acute Lung Injury in Mice.

Pathogen reduction technology (PRT) has been developed in an effort to make the blood supply safer, but there is controversy as to whether it may induce structural or functional changes to platelets that could lead to acute lung injury after transfusion. In this study, we used a commercial PRT system to treat human platelets that were then transfused into immunodeficient mice, and the development of acute lung injury was determined. P-selectin expression was higher in the Mirasol PRT-treated platelets compared to control platelets on storage day 5, but not storage day 1. Transfusion of control vs. Mirasol PRT-treated platelets (day 5 of storage, 109 platelets per mouse) into NOD/SCID mice did not result in lung injury, however transfusion of storage day 5 platelets treated with thrombin receptor-activating peptide increased both extravascular lung water and lung vascular permeability. Transfusion of day 1 platelets did not produce lung injury in any group, and LPS priming 24 hours before transfusion had no effect on lung injury. In a model of transfusion-related acute lung injury, NOD/SCID mice were susceptible to acute lung injury when challenged with H-2Kd monoclonal antibody vs. isotype control antibody. Using lung intravital microscopy, we did not detect a difference in the dynamic retention of platelets in the lung circulation in control vs. Mirasol PRT-treated groups. In conclusion, Mirasol PRT produced an increase in P-selectin expression that is storage-dependent, but transfusion of human platelets treated with Mirasol PRT into immunodeficient mice did not result in greater platelet retention in the lungs or the development of acute lung injury.

Targeting HSP90 Ameliorates Nephropathy and Atherosclerosis Through Suppression of NF-κB and STAT Signaling Pathways in Diabetic Mice.

Heat shock proteins (HSPs) are induced by cellular stress and function as molecular chaperones that regulate protein folding. Diabetes impairs the function/expression of many HSPs, including HSP70 and HSP90, key regulators of pathological mechanisms involved in diabetes complications. Therefore, we investigated whether pharmacological HSP90 inhibition ameliorates diabetes-associated renal damage and atheroprogression in a mouse model of combined hyperglycemia and hyperlipidemia (streptozotocin-induced diabetic apolipoprotein E-deficient mouse). Treatment of diabetic mice with 17-dimethylaminoethylamino-17-demethoxygeldanamycin (DMAG, 2 and 4 mg/kg, 10 weeks) improved renal function, as evidenced by dose-dependent decreases in albuminuria, renal lesions (mesangial expansion, leukocyte infiltration, and fibrosis), and expression of proinflammatory and profibrotic genes. Furthermore, DMAG significantly reduced atherosclerotic lesions and induced a more stable plaque phenotype, characterized by lower content of lipids, leukocytes, and inflammatory markers, and increased collagen and smooth muscle cell content. Mechanistically, the renoprotective and antiatherosclerotic effects of DMAG are mediated by the induction of protective HSP70 along with inactivation of nuclear factor-κB (NF-κB) and signal transducers and activators of transcription (STAT) and target gene expression, both in diabetic mice and in cultured cells under hyperglycemic and proinflammatory conditions. In conclusion, HSP90 inhibition by DMAG restrains the progression of renal and vascular damage in experimental diabetes, with potential implications for the prevention of diabetes complications.

Peptide-based inhibition of IκB kinase/nuclear factor-κB pathway protects against diabetes-associated nephropathy and atherosclerosis in a mouse model of type 1 diabetes.

AIMS/HYPOTHESIS: The canonical nuclear factor-κB (NF-κB) pathway mediated by the inhibitor of NF-κB kinase (IKK) regulates the transcription of inflammatory genes involved in the pathogenesis of diabetes, from the early phase to progression and final complications. The NF-κB essential modulator binding domain (NBD) contained in IKKα/ß is essential for IKK complex assembly. We therefore investigated the functional consequences of targeting the IKK-dependent NF-κB pathway in the progression of diabetes-associated nephropathy and atherosclerosis. METHODS: Apolipoprotein E-deficient mice with diabetes induced by streptozotocin were treated with a cell-permeable peptide derived from the IKKα/ß NBD region. Kidneys and aorta were analysed for morphology, leucocyte infiltrate, collagen, NF-κB activity and gene expression. In vitro studies were performed in renal and vascular cells. RESULTS: NBD peptide administration did not affect the metabolic severity of diabetes but resulted in renal protection, as evidenced by dose-dependent decreases in albuminuria, renal lesions (mesangial expansion, leucocyte infiltration and fibrosis), intranuclear NF-κB activity and proinflammatory and pro-fibrotic gene expression. Furthermore, peptide treatment limited atheroma plaque formation in diabetic mice by decreasing the content of lipids, leucocytes and cytokines and increasing plaque stability markers. This nephroprotective and anti-atherosclerotic effect was accompanied by a decline in systemic T helper 1 cytokines. In vitro, NBD peptide prevented IKK assembly/activation, p65 nuclear translocation, NF-κB-regulated gene expression and cell proliferation induced by either high glucose or inflammatory stimulation. CONCLUSIONS/INTERPRETATION: Peptide-based inhibition of IKK complex formation attenuates NF-κB activation, suppresses inflammation and retards the progression of renal and vascular injury in diabetic mice, thus providing a feasible approach against diabetes inflammatory complications.

Gene delivery of suppressors of cytokine signaling (SOCS) inhibits inflammation and atherosclerosis development in mice.

Chronic activation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway contributes to vascular inflammation and atherosclerosis by inducing expression of genes involved in cell proliferation, differentiation and migration. We aimed to investigate whether enforced expression of negative regulators, the suppressors of cytokine signaling (SOCS1 and SOCS3), inhibits harmful JAK/STAT-mediated responses and affects atherosclerosis in apolipoprotein E knockout mice. Adenovirus-mediated SOCS1 transgene expression impaired the onset and progression of atherosclerosis without impact on lipid profile, whereas SOCS3 was only effective on early atherosclerosis. Mechanistically, SOCS gene delivery, primarily SOCS1, attenuated STAT1 and STAT3 activation and reduced the expression of STAT-dependent genes (chemokine/chemokine receptors, adhesion molecules, pro-inflammatory cytokines and scavenger receptors) in aortic tissue. Furthermore, atherosclerotic plaques exhibit a more stable phenotype characterized by lower lipids, T cells and M1 macrophages and higher M2 macrophages and collagen. Atheroprotection was accompanied by a systemic alteration of T helper- and T regulatory-related genes and a reduced activation state of circulating monocytes. In vascular smooth muscle cells and macrophages, SOCS gene delivery inhibited cytokine-induced STAT activation, pro-inflammatory gene expression, cell migration and proliferation. In conclusion, targeting SOCS proteins, predominantly SOCS1, to suppress pathological mechanisms involved in atheroma plaque progression and destabilization could be an interesting anti-atherosclerotic strategy.

Neutrophil extracellular traps are pathogenic in primary graft dysfunction after lung transplantation.

RATIONALE: Primary graft dysfunction (PGD) causes early mortality after lung transplantation and may contribute to late graft failure. No effective treatments exist. The pathogenesis of PGD is unclear, although both neutrophils and activated platelets have been implicated. We hypothesized that neutrophil extracellular traps (NETs) contribute to lung injury in PGD in a platelet-dependent manner. OBJECTIVES: To study NETs in experimental models of PGD and in lung transplant patients. METHODS: Two experimental murine PGD models were studied: hilar clamp and orthotopic lung transplantation after prolonged cold ischemia (OLT-PCI). NETs were assessed by immunofluorescence microscopy and ELISA. Platelet activation was inhibited with aspirin, and NETs were disrupted with DNaseI. NETs were also measured in bronchoalveolar lavage fluid and plasma from lung transplant patients with and without PGD. MEASUREMENTS AND MAIN RESULTS: NETs were increased after either hilar clamp or OLT-PCI compared with surgical control subjects. Activation and intrapulmonary accumulation of platelets were increased in OLT-PCI, and platelet inhibition reduced NETs and lung injury, and improved oxygenation. Disruption of NETs by intrabronchial administration of DNaseI also reduced lung injury and improved oxygenation. In bronchoalveolar lavage fluid from human lung transplant recipients, NETs were more abundant in patients with PGD. CONCLUSIONS: NETs accumulate in the lung in both experimental and clinical PGD. In experimental PGD, NET formation is platelet-dependent, and disruption of NETs with DNaseI reduces lung injury. These data are the first description of a pathogenic role for NETs in solid organ transplantation and suggest that NETs are a promising therapeutic target in PGD.

Aspirin-triggered 15-epi-lipoxin A4 regulates neutrophil-platelet aggregation and attenuates acute lung injury in mice.

Evidence is emerging that platelets are major contributors to innate immune responses in conditions such as acute lung injury (ALI). Platelets form heterotypic aggregates with neutrophils, and we hypothesized that lipoxin mediators regulate formation of neutrophil-platelet aggregates (NPA) and that NPA significantly contribute to ALI. Lipopolysaccharide (LPS)-induced lung injury was accompanied by platelet sequestration, activation, intra-alveolar accumulation, and NPA formation within both blood and alveolar compartments. Using lung intravital microscopy, we observed the dynamic formation of NPA during physiologic conditions, which sharply increased with ALI. Aspirin (ASA) treatment significantly reduced lung platelet sequestration and activation, NPA formation, and lung injury. ASA treatment increased levels of ASA-triggered lipoxin (ATL; 15-epi-lipoxin A4), and blocking the lipoxin A4 receptor (ALX) with a peptide antagonist (Boc2) or using ALX knockouts (Fpr2/3(-/-)) reversed this protection. LPS increased NPA formation in vitro, which was reduced by ATL, and engagement of ALX by ATL on both neutrophils and platelets was necessary to prevent aggregation. In a model of transfusion-related acute lung injury (TRALI), Boc2 also reversed ASA protection, and treatment with ATL in both LPS and TRALI models protected from ALI. We conclude that ATL regulates neutrophil-platelet aggregation and that platelet-neutrophil interactions are a therapeutic target in lung injury.

Suppressor of cytokine signaling 1-derived peptide inhibits Janus kinase/signal transducers and activators of transcription pathway and improves inflammation and atherosclerosis in diabetic mice.

OBJECTIVE: Activation of Janus kinase/signal transducers and activators of transcription (STAT) pathway by hyperglycemia and dislypidemia contributes to the progression of diabetic complications, including atherosclerosis. Suppressor of cytokine signaling (SOCS) proteins negatively regulate Janus kinase/STAT and have emerged as promising target for anti-inflammatory therapies. We investigated whether a cell-permeable lipopeptide corresponding to the kinase inhibitory region of SOCS1 could reduce atherosclerosis in diabetic mice and identified the mechanisms involved. APPROACH AND RESULTS: Streptozotocin-induced diabetic apolipoprotein E-deficient mice (aged 8 and 22 weeks) were given intraperitoneal injections of vehicle, SOCS1-derived peptide, or control mutant peptide for 6 to 10 weeks. SOCS1 therapy suppressed STAT1/STAT3 activation in atherosclerotic plaques of diabetic mice and significantly reduced lesion size at both early and advanced stages of lesion development compared with vehicle group. Plaque characterization demonstrated that SOCS1 peptide decreased the accumulation of lipids, macrophages, and T lymphocytes, whereas increasing collagen and smooth muscle cell content. This atheroprotective effect was accompanied by systemic (reduced proinflammatory Ly6C(high) monocytes and splenic cytokine expression) and local (reduced aortic expression of chemokines and cytokines) mechanisms, without impact on metabolic parameters. In vitro, SOCS1 peptide dose dependently inhibited STAT1/STAT3 activation and target gene expression in vascular smooth muscle cells and macrophages and also suppressed cytokine-induced cell migration and adhesion processes. CONCLUSIONS: SOCS1-based targeting Janus kinase/STAT restrains key mechanisms of atherogenesis in diabetic mice, thereby preventing plaque formation and increasing plaque stability. Approaches to mimic native SOCS1 functions may have a therapeutic potential to retard the progression of diabetic complications.

Gene Deficiency in Activating Fcγ Receptors Influences the Macrophage Phenotypic Balance and Reduces Atherosclerosis in Mice.

Immunity contributes to arterial inflammation during atherosclerosis. Oxidized low-density lipoproteins induce an autoimmune response characterized by specific antibodies and immune complexes in atherosclerotic patients. We hypothesize that specific Fcγ receptors for IgG constant region participate in atherogenesis by regulating the inflammatory state of lesional macrophages. In vivo we examined the role of activating Fcγ receptors in atherosclerosis progression using bone marrow transplantation from mice deficient in γ-chain (the common signaling subunit of activating Fcγ receptors) to hyperlipidemic mice. Hematopoietic deficiency of Fcγ receptors significantly reduced atherosclerotic lesion size, which was associated with decreased number of macrophages and T lymphocytes, and increased T regulatory cell function. Lesions of Fcγ receptor deficient mice exhibited increased plaque stability, as evidenced by higher collagen and smooth muscle cell content and decreased apoptosis. These effects were independent of changes in serum lipids and antibody response to oxidized low-density lipoproteins. Activating Fcγ receptor deficiency reduced pro-inflammatory gene expression, nuclear factor-κB activity, and M1 macrophages at the lesion site, while increasing anti-inflammatory genes and M2 macrophages. The decreased inflammation in the lesions was mirrored by a reduced number of classical inflammatory monocytes in blood. In vitro, lack of activating Fcγ receptors attenuated foam cell formation, oxidative stress and pro-inflammatory gene expression, and increased M2-associated genes in murine macrophages. Our study demonstrates that activating Fcγ receptors influence the macrophage phenotypic balance in the artery wall of atherosclerotic mice and suggests that modulation of Fcγ receptor-mediated inflammatory responses could effectively suppress atherosclerosis.