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Crosstalk between metabolic and signaling events that induce tumor metastasis remains elusive. Here, we determine how oncogenic sphingosine 1-phosphate (S1P) metabolism induces intracellular C3 complement activation to enhance migration/metastasis. We demonstrate that increased S1P metabolism activates C3 complement processing through S1P receptor 1 (S1PR1). S1P/S1PR1-activated intracellular C3b-α′ 2 is associated with PPIL1 through glutamic acid 156 (E156) and aspartic acid 111 (D111) residues, resulting in NLRP3/inflammasome induction. Inactivation mutations of S1PR1 to prevent S1P signaling or mutations of C3b-α′ 2 to prevent its association with PPIL1 attenuate inflammasome activation and reduce lung colonization/metastasis in mice. Also, activation of the S1PR1/C3/PPIL1/NLRP3 axis is highly associated with human metastatic melanoma tissues and patient-derived xenografts. Moreover, targeting S1PR1/C3/PPIL1/NLRP3 signaling using molecular, genetic, and pharmacologic tools prevents lung colonization/metastasis of various murine cancer cell lines using WT and C3a-receptor1 knockout (C3aR1−/−) mice. These data provide strategies for treating high-grade/metastatic tumors by targeting the S1PR1/C3/inflammasome axis. [Display omitted] • Increased S1P metabolism activates C3 complement processing through S1PR1 • S1P/S1PR1-activated intracellular C3b-α′ 2 associates with PPIL1 inducing inflammasome • The S1PR1/C3/PPIL1/NLRP3 axis mediates cancer cell migration and tumor metastasis • Targeting S1PR1/C3/NLRP3 signaling prevents lung colonization/metastasis Janneh et al. define how sphingosine 1-phosphate (S1P) metabolism induces intracellular C3 complement activation to enhance cancer cell migration and metastasis via S1PR1/inflammasome signaling. Targeting the S1PR1/C3/inflammasome axis inhibits cancer cell migration/invasion and lung colonization, demonstrating the critical roles of the crosstalk between S1P metabolism and complement signaling in tumor metastasis. [ABSTRACT FROM AUTHOR]

Background Cigarette smoke (CS) plays a role in the exacerbation of chronic rhinosinusitis (CRS); however, the mechanism for this is unknown. We hypothesize that CS impairs human sinonasal epithelial cell (HSNEC) conversion of 25(OH)D3 (25VD3) to 1,25-dihydroxyvitamin D3 (1,25VD3) and, furthermore, that supplementation with 1,25VD3 will reverse smoke-induced inflammatory responses by HSNECs. Objective We sought to determine the effect of CS on vitamin D3 (VD3) levels, conversion, and regulation of CS-induced inflammation in control subjects and patients with CRS. Methods Blood and sinus tissue explants were collected at the time of surgery from control subjects, patients with chronic rhinosinusitis without nasal polyps, and patients with chronic sinusitis with nasal polyps (CRSwNP). Expression of VD3 metabolizing enzymes were measured by using RT-PCR. Primary HSNECs were cultured from tissue explants. 25VD3 with and without cigarette smoke extract (CSE) was used to examine conversion of 25VD3 to 1,25VD3, as well as HSNEC production of proinflammatory cytokines. Results CS exposure was associated with reduced circulating and sinonasal 25VD3 levels in all groups compared with those seen in CS-naive, disease-matched counterparts. CS exposure decreased expression of CYP27B1 and was especially pronounced in patients with CRSwNP. CSE impairs control HSNEC conversion of 25VD3. HSNECs from patients with CRSwNP also demonstrate an intrinsic reduction in conversion of 25VD3 to 1,25VD3. Exogenous 1,25VD3 reduces CSE-induced cytokine production by HSNECs. Conclusions Exposure to CS is associated with reduced 25VD3 levels and an impaired ability of HSNECs to convert 25VD3 to 1,25VD3. Addition of 1,25VD3 reduces the proinflammatory effects of CS on HSNECs. Impaired VD3 conversion by CS exposure represents a novel mechanism through which CS induces its proinflammatory effects. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice.To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD. [ABSTRACT FROM AUTHOR]

Background: Coronary artery vasculopathy (CAV) is the major life-limiting factor in cardiac transplantation, after 1 year. Antibody-mediated rejection (AMR) has been associated with development of both acute and chronic rejection. We analyzed endomyocardial biopsies for pathologic markers of AMR (C4d and C3d), from the first 2 years post-transplantation, to determine complement deposition in relation to the development of CAV. Methods: A retrospective, matched-pair study was used. Group 1 subjects (n = 26) were CAV-negative at 8 years, and Group 2 (n = 26) had angiographically detectable CAV at 4 years. Biopsies from six time-points were studied (total = 282). Immunohistochemistry was performed for C4d, C3d and CD68. Biopsies were graded for rejection using ISHLT criteria. Results: Although CAV was not significantly associated with C4d deposition, it was associated with C3d deposition (p = 0.043). Only 4% of C4d and 5% of C3d biopsies were completely negative. Group 1 had 6 AMR-positive biopsies, with Group 2 having 8. There was no significant relationship between acute cellular rejection or AMR events and CAV. Conclusions: This study demonstrates that complement deposition is a frequent occurrence in the first 2 years post-transplantation. Although acute rejection is a known risk factor for CAV, in this study the relationship was found not to be significant. No relationship was found with the development of CAV and histologic features of AMR, when assessed by C4d deposition alone. However, an association between C3d deposition and the development of CAV was determined in this study group, suggesting that complement activation may play a role in the pathogenesis of CAV. [Copyright &y& Elsevier]

Background: Acute rejection, a vascular-based disorder, has been identified as the major risk factor for obliterative bronchiolitis (OB), an airway-based pathology. This study investigated the hypothesis that changes to the microvascular blood supply of small airways were associated with the development of OB, thus providing a possible link between an acute vascular insult (acute rejection) and chronic airway changes (OB). Methods: Microvasculature of 695 small airways (99 patients) was assessed in post-mortem lung allograft specimens using monoclonal antibodies for von Willebrand factor and CD31. Group A consisted of 343 small airways from 58 patients with no evidence of OB. The remaining 41 patients had histological evidence of OB in some of their small airways and grouped as B, C, and D with some patients contributing to all three groups ie, their lung specimen had some small airways which were completely obliterated with OB, some airways which were partially obliterated and some small airways without any histological evidence of OB development. Thus group B consisted of 145 small airways (34 patients) without OB. Group C consisted of 171 small airways with partial luminal obstruction (36 patients). Group D consisted of 36 small airways (14 patients) with complete luminal obliteration. Results: Airway circumference (mean ± standard deviation) was 2.36 ± 0.37, 2.41 ± 0.51, 2.49 ± 0.51, and 2.57 ± 0.79 mm, respectively (p = 0.40). Mean number of blood vessels per unit length of airway circumference was 4.12 ± 1.1, 1.58 ± 0.61, 2.42 ± 1.06, and 4.42 ± 1.46 vessels/mm, respectively (p < 0.001). Blood vessels with circumference greater than 0.2 mm were present in 100%, 64%, 39%, and 7% of small airways, respectively (p < 0.001). Univariate and multivariate analyses (donor and recipient age, sex, and cytomegalovirus status, recipient pretransplant diagnosis, ischemic times, acute rejection and infective episodes, postoperative survival days, recipient group [A to D], blood vessels per unit length, and airway circumference) confirmed that reduction in blood vessels per unit length was associated with the development of OB and was time-independent. Conclusions: Obliterative bronchiolitis was preceded by a decrease in microvascular supply to the small airways (group B). The subsequent onset of airway scarring (groups C and D) was associated with an increased number of significantly smaller vessels, suggestive of neovascularization. [Copyright &y& Elsevier]

Background: Endothelial activation in the donor heart has been described variably after brain death and transplantation. We aimed to characterize the time course of endothelial activation in right ventricle (RV) and left ventricle (LV) during the acute phase of clinical transplantation. Methods: We studied biopsy specimens from the RVs and the LVs of 40 donor hearts: at initial assessment of the donor, at end-ischemia, and after 10 minutes of reperfusion. We also included follow-up RV biopsy specimens at 1 week, 1 month, and 3 months after surgery. Six of the patients had cystic fibrosis and were domino donors. Results: P-selectin and vascular cell adhesion molecule 1 (VCAM-1), but not E-selectin were up-regulated in brain-dead and in domino donors vs controls. Unused donor hearts (n = 6) had significantly less up-regulation of P-selectin and of VCAM-1. We found no difference between the RV and the LV during surgery, but we did see important time-dependent variations. P-selectin was present in 85% of vessels throughout transplantation and decreased to approximately 60% after transplantation (p < 0.001). We initially detected VCAM-1 in 20% of vessels, which decreased to 5% during storage, then increased to 47% at reperfusion, and gradually decreased thereafter (p < 0.001). E-selectin expression increased progressively from 15% initially to 45% at reperfusion and then decreased after surgery (p = 0.001). Thrombomodulin expression was decreased at baseline, and the decrease was accentuated afterward (p = 0.02). Patients with donor organ failure did not have a specific pattern of endothelial activation. Conclusion: Cardiac transplantation is associated with marked endothelial activation, with no difference between the two ventricles. The changes persist in the post-operative period, even in the absence of acute rejection. [Copyright &y& Elsevier]

Background: Vascular remodeling is central to the development of transplant coronary artery vasculopathy (CAV). For remodeling to occur, a sustained blood and nutrient supply is essential. Here we report on the presence of angiogenesis within the neointima of coronary arteries from cardiac transplant recipients. Methods: Coronary arteries from 57 cardiac transplant recipients with CAV were analyzed. Immunocytochemistry with antibodies raised against endothelial cells (CD31, CD34, and vWF), vascular smooth muscle cells (SmA), and activated endothelial cells (MHC 2, P-SEL, E-SEL, and VCAM-1) was performed. Results: A total of 89% of patients had significant angiogenesis. These vessels appeared as endothelial lined channels and were present in a concentric circumferential pattern within the mid portion of the neointima. These new vessels were present at an interface between an area of intimal hyperplasia and below an area of fibrous regeneration. These 2 distinct zones were present in 64% of the cases, and were clearly demonstrated with an elastic van Gieson (EVG) stain and are distinctly different from that seen in native atherosclerosis. Endothelial activation markers were strongly expressed by the endothelial cells lining new vessels, suggesting that they are functional and may aid in the recruitment of inflammatory cells. Conclusions: These data suggest that angiogenesis is present within the intima of CAV lesions and may contribute to the continued obliteration of the vessel lumen. The vessels appear to originate in the intima and may represent the location of the donor endothelium before transplantation. Inhibition of endothelial damage may provide therapeutic options to prevent the progression of CAV. [Copyright &y& Elsevier]

: BackgroundTransplant coronary artery vasculopathy (CAV) is a fibro-proliferative process that leads to lumen occlusion and cardiac failure. Current theories suggest that the process evolves over time in response to inflammation and proliferation of donor derived medial smooth muscle cells (SMC). Animal models of cardiac transplantation have suggested that the neointima is formed by recipient derived circulating progenitor cells. The aim of this investigation is to determine the origin of the neointimal SMC within epicardial coronary arteries from human cardiac allografts by using sex mis-matched recipients and donors, and a Y specific chromosome probe.: MethodsCoronary arteries from 14 patients previously assessed histologically to have CAV were analyzed—eight male recipients of female donor organs, 2 female-to-female, and 4 male-to-male transplants. A double immunocytochemistry and in-situ hybridization technique using a Y chromosome DNA probe and either antibodies to smooth muscle actin or Ham-56 a macrophage marker were employed.: ResultsNo Y chromosome bodies could be identified in the female-to-female allografts. In the 4 male donor and male recipient cases, cells positive for the Y chromosome probe were identified. In sex mis-matched transplants, female to male, inflammatory cells marked with Ham-56 were also positive for Y chromosome probe. Flattened cells positive for Y chromosome were observed just beneath the endothelial surface. When double stained, these were identified as infiltrating macrophages. No double staining smooth muscle cells and Y chromosome positive cells could be identified within the neointima.: ConclusionsThis study confirms the source of SMC of the neointima of CAV lesions from epicardial coronary arteries to be of donor origin. In contrast to animal models, circulating progenitor cells do not appear to play a role within the neointima of human transplant CAV. [Copyright &y& Elsevier]

Dysregulation of inflammation-resolution pathways leads to postlung transplant (LTx) ischemia-reperfusion (IR) injury and allograft dysfunction. Our hypothesis is that combined treatment with specialized pro-resolving lipid mediators, that is, Resolvin D1 (RvD1) and Maresin-1 (MaR1), enhances inflammation-resolution of lung IR injury. Expression of RvD1 and MaR1 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on days 0, 1, and 7 post-LTx. Lung IR injury was evaluated in C57BL/6 (WT), FPR2−/−, and LGR6 siRNA treated mice using a hilar-ligation model with or without administration with RvD1 and/or MaR1. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by RvD1 and MaR1 against lung IR injury. I n vitro studies analyzed alveolar macrophages and type II epithelial cell activation after treatment with RvD1 or MaR1. RvD1 and MaR1 expressions in BAL from post-LTx patients was significantly increased on day 7 compared to days 0 and 1. Concomitant RvD1 and MaR1 treatment significantly mitigated early pulmonary inflammation and lung IR injury in WT mice, which was regulated via FPR2 and LGR6 receptors. In the murine orthotopic donation after cardiac death LTx model, RvD1 and MaR1 treatments significantly attenuated lung IR injury and increased PaO 2 levels compared to saline-treated controls. Mechanistically, RvD1/FPR2 signaling on alveolar macrophages attenuated HMGB1 and TNF-α secretion and upregulated uptake of macrophage-dependent apoptotic neutrophils (efferocytosis), whereas MaR1/LGR6 signaling mitigated CXCL1 secretion by epithelial cells. Bioactive proresolving lipid mediator-dependent signaling that is, RvD1/FPR2 and MaR1/LGR6- offers a novel therapeutic strategy in post-LTx injury. [ABSTRACT FROM AUTHOR]

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. The pathogenesis of COPD is complex; however, recent studies suggest autoimmune changes, characterized by the presence of autoantibodies to elastin and collagen, may contribute to disease status. COPD patients make up approximately 30% of all lung transplants (LTx) annually, however, little is known regarding the relationship between COPD-related autoantibodies and LTx outcomes. We hypothesized that COPD patients that undergo LTx and develop primary graft dysfunction (PGD) have altered circulating autoantibody levels and phenotypic changes as compared those COPD-LTx recipients that do not develop PGD. We measured total immunoglobulin and circulating elastin and collagen autoantibody levels in a cohort of COPD lung transplant recipients pre- and post-LTx. No significant differences were seen in total, elastin, or collagen IgM, IgG, IgG1, IgG2, IgG3, and IgG4 antibodies between PGD+ and PGD- recipients. Antibody function can be greatly altered by glycosylation changes to the antibody Fc region and recent studies have reported altered IgG glycosylation profiles in COPD patients. We therefore utilized a novel mass spectrometry-based multiplexed N-glycoprotein imaging approach and measured changes in IgG-specific antibody N-glycan structures. COPD-LTx recipients who developed PGD had significantly increased IgG1 N-glycan signatures as compared PGD- recipients. In conclusion, we show that immunoglobulin and autoreactive antibody levels are not significantly different in COPD LTx recipients that develop PGD. However, using a novel IgG glycomic analysis we were able to demonstrate multiple significant increases in IgG1 specific N-glycan signatures that were predictive of PGD development. Taken together, these data represent a potential novel method for identifying COPD patients at risk for PGD development and may provide clues to mechanisms by which antibody N-glycan signatures could contribute to antibody-mediated PGD pathogenesis. [ABSTRACT FROM AUTHOR]

Post-transplant ischemia reperfusion injury (IRI) is a recognized risk factor for subsequent organ dysfunction, alloresponsiveness, and rejection. The complement system is known to play a role in IRI and represents a therapeutic target. Complement is activated in transplanted grafts when circulating IgM antibodies bind to exposed ischemia-induced neoepitopes upon reperfusion, and we investigated the targeting of a human complement inhibitor, CR1, to a post-transplant ischemia-induced neoepitope. A fragment of human CR1 was linked to a single chain antibody construct (C2 scFv) recognizing an injury-specific neoepitope to yield C2-CR1. This construct, along with a soluble untargeted counterpart, was characterized in a cardiac allograft transplantation model of IRI in terms of efficacy and safety. CR1 was similarly effective against mouse and human complement. C2-CR1 provided effective protection against cardiac IRI at a lower dose than untargeted CR1. The increased efficacy of C2-CR1 relative to CR1 correlated with decreased C3 deposition, and C2-CR1, but not CR1, targeted to cardiac allografts. At a dose necessary to reduce IRI, C2-CR1 had minimal impact on serum complement activity, in contrast to CR1 which resulted in a high level of systemic inhibition. The circulatory half-life of CR1 was markedly longer than that of C2-CR1, and whereas a minimum therapeutic dose of CR1 severely impaired host susceptibility to infection, C2-CR1 had no impact. We show the translational potential of a human complement inhibitor targeted to a universal ischemia-induced graft-specific epitope, and demonstrate advantages compared to an untargeted counterpart in terms of efficacy and safety. [ABSTRACT FROM AUTHOR]

Our hypothesis is that the immunomodulatory capacities of mesenchymal stem cell‒derived extracellular vesicles (EVs) can be enhanced by specific microRNAs (miRNAs) to effectively attenuate post-transplant lung ischemia‒reperfusion (IR) injury. The expression of miR-206 was analyzed in bronchoalveolar lavage (BAL) fluid of patients on Days 0 and 1 after lung transplantation. Lung IR injury was evaluated in C57BL/6 mice using a left lung hilar-ligation model with or without treatment with EVs or antagomiR-206‒enriched EVs. Murine lung tissue was used for miRNA microarray hybridization analysis, and cytokine expression, lung injury, and edema were evaluated. A donation after circulatory death and murine orthotopic lung transplantation model was used to evaluate the protection by enriched EVs against lung IR injury. In vitro studies analyzed type II epithelial cell activation after coculturing with EVs. A significant upregulation of miR-206 was observed in the BAL fluid of patients on Day 1 after lung transplantation compared with Day 0 and in murine lungs after IR injury compared with sham. Treatment with antagomiR-206‒enriched EVs attenuated lung dysfunction, injury, and edema compared with treatment with EVs alone after murine lung IR injury. Enriched EVs reduced lung injury and neutrophil infiltration as well as improved allograft oxygenation after murine orthotopic lung transplantation. Enriched EVs significantly decreased proinflammatory cytokines, especially epithelial cell‒dependent CXCL1 expression, in the in vivo and in vitro IR injury models. EVs can be used as biomimetic nanovehicles for protective immunomodulation by enriching them with antagomiR-206 to mitigate epithelial cell activation and neutrophil infiltration in the lungs after IR injury. [ABSTRACT FROM AUTHOR]

: Background:The use of pig organs transgenic for human decay accelerating factor (hDAF) has largely overcome the problems of hyperacute rejection. With improved immunosuppressive protocols, life supporting grafts are showing greater survival times bringing the possibility of clinical xenotransplantation closer. Examination of the histopathology of the rejection process provides insight into the underlying mechanism and may suggest ways in which new immunosuppressive strategies should be directed.: Methods:44 baboons (Papio anubis) underwent heart transplants of which 39 were from transgenic donors. The transplanted organs were examined histologically and stained for evidence of immunoglobulin and complement deposition as well as cellular infiltrates.: Results:In the transgenic animals survival times were 2 to 99 days (mean 23.5) and the heterotopic group and 1 to 39 days (mean 11.7) in the orthotopic group. There were 3 cases of hyperacute rejection between the 2 groups. Rejected organs showed areas of old and recent myocardial infarction associated with vascular thrombosis. There was widespread deposition within vessels of immunoglobulins IgM and IgG together with complement fractions C3 and C5b to 9 in those organs that were rejected. The amount of complement positive in the longer surviving organs was less than those rejecting early. Cellular infiltate was predominantly macrophage with some later appearing T or natural killer cells.: Conclusions:The histopathological changes support the importance of immunoglobulin and complement in delayed xenograft or acute vascular rejection. With time there is an increase in cellular infiltrate predominantly macrophages and these findings suggest an increasingly important role for the cells and the rejection process. The presence of areas of infarction and underlying vascular thrombosis is in keeping with endothelial activation and the establishment of procoagulant phenotype which may be due to immunoglobulin, complement, secreted cytokines and direct cellular effects. [Copyright &y& Elsevier]