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Purpose Polysorbate excipients are commonly used as surfactants to stabilize therapeutic proteins in formulations. Degradation of polysorbates could lead to particle formation and instability of the drug formulation. We investigated how the fatty acid composition of polysorbate 80 impacts the degradation profile, particle formation, and product stability under stress conditions. Methods Two polysorbate 80-containing therapeutic protein formulations were reformulated with either Polysorbate 80 NF synthesized from a fatty acid mixture that contains mainly oleic acid (≥58%) or a version of polysorbate 80 synthesized with high oleic acid (>98%). Stress conditions, including high temperature and esterase spiking, were applied and changes to both the polysorbate and the therapeutic protein product were investigated for stability, purity, innate immune response and biological activity. Results The addition of esterase and storage at 37°C led to significant hydrolysis of the polysorbate and increases in sub-visible particle formation for both polysorbates tested. The fatty acid composition of polysorbate 80 did not directly alter the stability profile of either therapeutic protein as measured by size exclusion chromatography, or significantly impact innate immune response or biological activity. However, formulations with Polysorbate 80 NF showed greater propensity for sub-visible particle formation under stress conditions. Conclusions These results suggest that composition of fatty acids in polysorbate 80 may be a promoter for sub-visible particulate formation under the stress conditions tested but may not impact protein aggregation or biological activity.

Objective Dysregulation of innate and adaptive immune responses contributes to the pathogenesis of systemic lupus erythematosus (SLE) and its associated premature vascular damage. No drug to date targets both systemic inflammatory disease and the cardiovascular complications of SLE. Tofacitinib is a JAK inhibitor that blocks signaling downstream of multiple cytokines implicated in lupus pathogenesis. While clinical trials have shown that tofacitinib exhibits significant clinical efficacy in various autoimmune diseases, its role in SLE and the associated vascular pathology remains to be characterized. Methods MRL/lpr lupus-prone mice were administered tofacitinib or vehicle by gavage for 6 weeks (therapeutic arm) or 8 weeks (preventive arm). Nephritis, skin inflammation, serum levels of autoantibodies and cytokines, mononuclear cell phenotype and gene expression, neutrophil extracellular traps (NETs) release, endothelium-dependent vasorelaxation, and endothelial differentiation were compared in treated and untreated mice. Results Treatment with tofacitinib led to significant improvement in measures of disease activity, including nephritis, skin inflammation, and autoantibody production. In addition, tofacitinib treatment reduced serum levels of proinflammatory cytokines and interferon responses in splenocytes and kidney tissue. Tofacitinib also modulated the formation of NETs and significantly increased endothelium-dependent vasorelaxation and endothelial differentiation. The drug was effective in both preventive and therapeutic strategies. Conclusion Tofacitinib modulates the innate and adaptive immune responses, ameliorates murine lupus, and improves vascular function. These results indicate that JAK inhibitors have the potential to be beneficial in SLE and its associated vascular damage.

Cutaneous leishmaniasis (CL) affects the lives of 0.7–1 million people every year causing lesions that take months to heal. These lesions can result in disfiguring scars with psychological, social and economic consequences. Antimonials are the first line of therapy for CL, however the treatment is lengthy and linked to significant toxicities; further, its efficacy is variable and resistant parasites are emerging. Shorter or lower dose antimonial treatment regimens, which would decrease the risk of adverse events and improve patient compliance, have shown reduced efficacy and further increase the risk emergence of antimonial-resistant strains. The progression of lesions in CL is partly determined by the immune response it elicits, and previous studies showed that administration of immunomodulatory type D CpG ODNs, magnifies the immune response to Leishmania and reduces lesion severity in nonhuman primates (NHP) challenged with Leishmania major or Leishmania amazonensis. Here we explored whether the addition of a single dose of immunomodulating CpG ODN D35 augments the efficacy of a short-course, low-dose pentavalent antimonial treatment regimen. Results show that macaques treated with D35 plus 5mg/kg sodium stibogluconate (SbV) for 10 days had smaller lesions and reduced time to re-epithelization after infection with Leishmania major. No toxicities were evident during the studies, even at doses of D35 10 times higher than those used in treatment. Critically, pentavalent antimonial treatment did not modify the ability of D35 to induce type I IFNs. The findings support the efficacy of D35 as adjuvant therapy for shorter, low dose pentavalent antimonial treatment., Author summary Cutaneous leishmaniasis is a devastating disease that affects close to a million people every year. Its clinical presentation ranges from small uncomplicated lesions that heal over a few months to debilitating large chronic or recurring lesions that result in disfigurement, stigma, and economic loss. Antimonials are the first line treatment for cutaneous leishmaniasis in most countries, but the lengthy treatment schedules, significant associated toxicities, and the emergence of resistant strains, require the development of alternative strategies. As the immune response is a key determinant of disease course, immunomodulatory therapies could be harnessed to act in concert with antimonials to improve the safety and efficacy of CL treatment. Synthetic oligonucleotide D35 selectively activates plasmacytoid dendritic cells and was previously shown to reduce the severity of L. major and L. amazonensis lesions in rhesus macaques, but its activity in combination with antimonials was unknown. Our studies show that a single subcutaneous dose of innate immune modulator D35 improved the response to a low-dose abbreviated antimonial course, reducing the severity of the lesions and accelerating healing in primates. No toxicities were evident with D35 at doses ten-fold higher than the effective dose. The studies suggest that the combined therapy strategy shows clinical promise.

We report a synthetic route to BODIPY-cholesterol conjugates in which the key steps are Suzuki or Liebeskind-Srogl cross-coupling of cholesterol phenyl moieties 11, 12, and 13 with structurally diverse BODIPY scaffolds. All conjugates feature single-bonded and hydrophobic linkages between the fluorophore and sterol devoid of heteroatoms. Using HeLa cells, we show that these BODIPY-cholesterol analogs can be used simultaneously with the parent BODIPY-cholesterol for cell imaging and flow cytometry. The BODIPY-cholesterol analogs exhibit a similar cellular localization in HeLa cells and show similar cholesterol efflux properties from THP1 cells to HDL acceptors. These results demonstrate that the red-shifted BODIPY-cholesterol analogs behave in a manner similar to unlabeled cholesterol and are useful probes for simultaneous visualization of intracellular cholesterol pools and for monitoring cholesterol efflux from cells to extracellular acceptors.

Objective— Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood. Approach and Results— Septic stress induces glucocorticoids production which triggers thymocyte apoptosis. Here, we used scavenger receptor BI (SR-BI)–null mice, which are completely deficient in inducible glucocorticoids in sepsis, to investigate the regulation of thymocyte apoptosis in sepsis. Cecal ligation and puncture induced profound thymocyte apoptosis in SR-BI +/+ mice, but no thymocyte apoptosis in SR-BI −/− mice because of lack of inducible glucocorticoids. Unexpectedly, supplementation of glucocorticoids only partly restored thymocyte apoptosis in SR-BI −/− mice. We demonstrated that high-density lipoprotein (HDL) is a critical modulator for thymocyte apoptosis. SR-BI +/+ HDL significantly enhanced glucocorticoid-induced thymocyte apoptosis, but SR-BI −/− HDL had no such activity. Further study revealed that SR-BI +/+ HDL modulates glucocorticoid-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI −/− HDL loses such regulatory activity. To understand why SR-BI −/− HDL loses its regulatory activity, we analyzed HDL cholesterol contents. There was 3-fold enrichment of unesterified cholesterol in SR-BI −/− HDL compared with SR-BI +/+ HDL. Normalization of unesterified cholesterol in SR-BI −/− HDL by probucol administration or lecithin cholesteryl acyltransferase expression restored glucocorticoid-induced thymocyte apoptosis, and incorporating unesterified cholesterol into SR-BI +/+ HDL rendered SR-BI +/+ HDL dysfunctional. Using lckCre-GR fl/fl mice in which thymocytes lack cecal ligation and puncture–induced thymocyte apoptosis, we showed that lckCre-GR fl/fl mice were significantly more susceptible to cecal ligation and puncture–induced septic death than GR fl/fl control mice, suggesting that glucocorticoid-induced thymocyte apoptosis is required for protection against sepsis. Conclusions— The findings in this study reveal a novel regulatory mechanism of thymocyte apoptosis in sepsis by SR-BI and HDL.

Aspirin (ASA) is known to alter the production of potent inflammatory lipid mediators, but whether it interacts with omega-3 fatty acids (FAs) from fish oil to affect atherosclerosis has not been determined. The goal was to investigate the impact of a fish oil-enriched diet alone and in combination with ASA on the production of lipid mediators and atherosclerosis. ApoE(-/-) female mice were fed for 13weeks one of the four following diets: omega-3 FA deficient (OD), omega-3 FA rich (OR) (1.8g omega-3 FAs/kg·diet per day), omega-3 FA rich plus ASA (ORA) (0.1g ASA/kg·diet per day) or an omega-3 FA deficient plus ASA (ODA) with supplement levels equivalent to human doses. Plasma lipids, atherosclerosis, markers of inflammation, hepatic gene expression and aortic lipid mediators were determined. Hepatic omega-3 FAs were markedly higher in OR (9.9-fold) and ORA (7-fold) groups. Mice in both OR and ORA groups had 40% less plasma cholesterol in very low-density lipoprotein-cholesterol and low-density lipoprotein fractions, but aortic plaque area formation was only significantly lower in the ORA group (5.5%) compared to the OD group (2.5%). Plasma PCSK9 protein levels were approximately 70% lower in the OR and ORA groups. Proinflammatory aortic lipid mediators were 50%-70% lower in the ODA group than in the OD group and more than 50% lower in the ORA group. In summary, less aortic plaque lesions and aortic proinflammatory lipid mediators were observed in mice on the fish oil diet plus ASA vs. just the fish oil diet.

Familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, and chemical properties, to wild-type and Lcat -/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat -/- mice, which have low HDL but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat -/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes, where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA 2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat -/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of LCAT induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.

Interleukin-1β (IL-1β), a potent pro-inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL-1β is controlled by a multi-protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High-density lipoprotein (HDL) has consistently been shown to be anti-atherogenic and to have anti-inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL-1β secretion in response to cholesterol crystals in THP-1 cells and in human-monocyte-derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro-IL-1β and NLRP3, and reducing caspase-1 activation. In summary, we have described a novel anti-inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.

Systemic lupus erythematosus (SLE) is an autoimmune disease with heterogeneous manifestations including severe organ damage and vascular dysfunction leading to premature atherosclerosis. IFN-α has been proposed to have an important role in the development of lupus and lupus-related cardiovascular disease, partly by repression of IL-1 pathways leading to impairments in vascular repair induced by endothelial progenitor cells (EPCs) and circulating angiogenic cells (CACs). Counterintuitively, SLE patients also display transcriptional upregulation of the IL-1β/IL-18 processing machinery, the inflammasome. To understand this dichotomy and its impact on SLE-related cardiovascular disease, we examined cultures of human and murine control or lupus EPC/CACs to determine the role of the inflammasome in endothelial differentiation. We show that caspase-1 inhibition improves dysfunctional SLE EPC/CAC differentiation into mature endothelial cells and blocks IFN-α–mediated repression of this differentiation, implicating inflammasome activation as a crucial downstream pathway leading to aberrant vasculogenesis. Furthermore, serum IL-18 levels are elevated in SLE and correlate with EPC/CAC dysfunction. Exogenous IL-18 inhibits endothelial differentiation in control EPC/CACs and neutralization of IL-18 in SLE EPC/CAC cultures restores their capacity to differentiate into mature endothelial cells, supporting a deleterious effect of IL-18 on vascular repair in vivo. Upregulation of the inflammasome machinery was operational in vivo, as evidenced by gene array analysis of lupus nephritis biopsies. Thus, the effects of IFN-α are complex and contribute to an elevated risk of cardiovascular disease by suppression of IL-1β pathways and by upregulation of the inflammasome machinery and potentiation of IL-18 activation.

Individuals with systemic lupus erythematosus (SLE) have a striking increase in the risk of premature atherosclerosis, a complication preceded by significant subclinical vascular damage. A proposed mechanism leading to accelerated vascular disease in SLE is an imbalance between vascular damage and repair, as patients with this disease display significant abnormalities in phenotype and function of endothelial progenitor cells. In addition, individuals with SLE have a higher incidence of insulin resistance which may further contribute to the increased cardiovascular risk. This study examined the role of the peroxisome proliferator activated receptor γ agonist pioglitazone in improving endothelial function, endothelial progenitor cell numbers and functional capacity, metabolic parameters, and disease activity in the lupus-prone murine model New Zealand Black/New Zealand White (NZB × NZW)F1. Ten-week-old prenephritic female NZB/NZW F1 mice were exposed to 10 or 25 mg/kg/day of oral pioglitazone or vehicle for 15 or 24 wk. Mice exposed to pioglitazone exhibited pronounced enhancement in endothelial-dependent vasorelaxation of thoracic aortas and in endothelial progenitor cell function, as assessed by the capacity of bone marrow-derived endothelial progenitor cells to differentiate into mature endothelial cells. Pioglitazone-treated mice showed improvement in insulin resistance, adipokine, and lipid profile. Kidneys from pioglitazone-treated mice showed significant decreases in immune complex deposition, renal inflammation, T cell glomerular infiltration, and intrarenal synthesis of TNF-α, IL-1β, and VCAM-1. These results indicate that peroxisome proliferator-activated receptor γ agonists could serve as important tools in the prevention of premature cardiovascular disease and organ damage in SLE.

Individuals with systemic lupus erythematosus (SLE) have a striking increase in premature atherosclerosis of unclear etiology. Accelerated endothelial cell apoptosis occurs in SLE and correlates with endothelial dysfunction. Endothelial progenitor cells (EPCs) and myelomonocytic circulating angiogenic cells (CACs) are crucial in blood vessel repair after vascular damage, and decreased levels or abnormal function of EPCs/CACs are established atherosclerosis risk factors. We investigated if vascular repair is impaired in SLE. We report that SLE patients display abnormal phenotype and function of EPCs/CACs. These abnormalities are characterized by significant decreases in the number of circulating EPCs (310 ± 50 EPCs/mL of blood in SLE versus 639 ± 102 in controls) and significant impairments in the capacity of EPCs/CACs to differentiate into mature ECs and synthesize adequate levels of the proangiogenic molecules vascular endothelial growth factor (VEGF) and hepatic growth factor (HGF). These abnormalities are triggered by interferon-α (IFN-α), which induces EPC and CAC apoptosis and skews myeloid cells toward nonangiogenic phenotypes. Lupus EPCs/CACs have increased IFN-α expression and their supernatants promote higher induction of IFN-inducible genes. Importantly, neutralization of IFN pathways restores a normal EPC/CAC phenotype in lupus. SLE is characterized by an imbalance between endothelial cell damage and repair triggered by type I IFNs, which might promote accelerated atherosclerosis.

Improved understanding of the effects of dairy-waste treatment and land application on microbial processes and products is required to predict the outcome of waste applications and avoid undesirable environmental impacts. Our objective was to assess effects of treated dairy-waste on soil N pools, nitrification, plant N availability, and yield in a silage cornfield (Zea mays L.) treated with ammonium sulfate (AS), dairy-waste compost (DC), or liquid dairy-waste (LW) as N sources at two levels of application over 5 yr. Increases in soil C and N, nitrate, and available P and K were observed for the DC treated soils throughout the 5-yr period. Soil organic C increases for the high-level DC treated soil doubled the C pool resulting in an increase of 14 Mg C ha 21 . The highest nitrate accumulation was at the 60- to 90-cm depth for soils receiving high level of DC (200 kg N ha 21 ), which moved to lower depths in subsequent years. Soils receiving a high-level of DC or LW showed a three-fold increase in nitrifier activity compared with the control. There was a positive silage corn yield response with all the treatments, with DC having the highest yields. While N from AS and LWare available for plant uptake almost immediately, the organic N in compost continued to mineralize throughout the growing season, after harvest and in subsequent years. Careful management of application rates to optimize the timing of N release versus plant demand and of post-harvest nutrient pools are suggested for the prevention of excessive nitrate accumulations and movement from repeated dairywaste applications.

Measurements of gross N transformation rates are important to properly understand N cycling processes in agricultural soils where both productive and consumptive processes occur. The objective of the study was to determine the effect of repeated application of dairywaste compost (DC), liquid dairy-waste (LW), and ammonium sulfate (AS) on gross N mineralization and nitrification rates and N supplying potential of an agricultural soil. Our goal was to examine both production and consumption of inorganic N for their effects on the balance between N supply from treated dairy-wastes and plant N demand. Treatments were applied at rates approximately equivalent to 100 and 200 kg available N ha 21 for 6 yr annually. Field-based N 15 pool dilution techniques and laboratory incubation experiments were employed to measure gross rates and mineralization potential of the soil. Both levels of DC raised the labile organic N pool significantly but only the high level DC significantly increased the decomposition rate constant (k). The mean gross N mineralization rates for 1999 to 2002 for the high levels of DC, LW, and AS were 5.72, 2.89, and 1.27 mg N kg 21 d 21 , whereas gross nitrification rates were 10.24, 1.57, and 0.74 mg N kg 21 d 21 , respectively. Net mineralization rates were ,35% of gross rates while nitrate consumption was not significant under any treatment. Variability in gross rates was high in the soils receiving DC, which could be due to presence of hotspots of labile organic matter. Elevated gross N transformation rates in plots receiving DC indicate the dynamic nature of this agricultural soil after repeated applications of dairy-waste.

cope α-Cyclodextrin (α-CD), a cyclic polymer of glucose, has been shown to lower plasma cholesterol in animals and humans; however, its effect on atherosclerosis has not been previously described. Methods and results apoE-knockout mice were fed either low-fat diet (LFD; 5.2% fat, w/w), or Western high fat diet (21.2% fat) containing either no additions (WD), 1.5% α-CD (WDA); 1.5% β-CD (WDB); or 1.5% oligofructose-enriched inulin (WDI). Although plasma lipids were similar after 11 weeks on the WD vs. WDA diets, aortic atherosclerotic lesions were 65% less in mice on WDA compared to WD (P < 0.05), and similar to mice fed the LFD. No effect on atherosclerosis was observed for the other WD supplemented diets. By RNA-seq analysis of 16S rRNA, addition of α-CD to the WD resulted in significantly decreased cecal bacterial counts in genera Clostridium and Turicibacterium, and significantly increased Dehalobacteriaceae. At family level, Comamonadaceae significantly increased and Peptostreptococcaceae showed a negative trend. Several of these bacterial count changes correlated negatively with % atherosclerotic lesion and were associated with increased cecum weight and decreased plasma cholesterol levels. Conclusion Addition of α-CD to the diet of apoE-knockout mice decreases atherosclerosis and is associated with changes in the gut flora. This article is protected by copyright. All rights reserved

High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223(-/-) mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDL's anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells.

Introduction Free cholesterol plays a vital role in maintaining cell membrane fluidity and function. Elevated levels of cholesterol in red blood cells (RBC) and platelets have been demonstrated to impair the function of these cells. In circulation, free cholesterol freely exchanges between cells and lipoproteins and lecithin-cholesterol acyltransferase (LCAT) plays a role in this exchange. The esterification of cholesterol by LCAT sequesters it in the interior of lipoprotein particles and thereby limits its movement between lipoproteins and cells. Previously, SR-B1 KO mice have been shown to have dysfunctional RBCs and platelets and this dysfunction is exacerbated by a high fat diet. These mice have a partial defect in cholesterol esterification and are susceptible to diet induced atherosclerosis. Hypothesis We assessed in SR-BI KO mice whether increased cholesterol esterification by LCAT would improve numbers of RBCs and platelets, as well as prevent the development of atherosclerosis. Methods and Results Control (C57BL/6), SR-B1 KO, SR-B1 KO x LCAT KO(DKO), and SR-B1 KO x LCAT-Trg(LCAT-Trg) mice were maintained on a western diet for 7 months. As previously observed, SR-B1 KO mice had decrease numbers of RBCs and platelets compared to control mice, and over expression of LCAT restored levels to that of wild type mice, while loss of LCAT further abrogated the decrease in cell counts(n=10). Unexpectedly, LCAT-Trg mice has the highest levels of cholesterol in plasma, but RBC and platelets showed the lowest levels of free cholesterol, as assessed by filipin staining. Atherosclerosis in western diet fed mice was assessed by Oil Red O in two ways by looking at the aortic root(n=4) and by en face analysis of the aorta(n=10). In the aortic root SRB-1KO and DKO mice showed 3x the amount of lesion size as controls, while LCAT-Trg mice had levels comparable to the controls mice. The en face analysis also showed that DKO mice had 3x the amount of lesions, while LCAT-Trg and SRB-1KO mice had levels comparable to control mice. Conclusion LCAT is able to modulate the movement of cholesterol between circulating cells and lipoproteins thereby protecting RBCs and platelets. Our results further demonstrate a possible benefit of LCAT on the progression of atherosclerosis.

Human familial lecithin:cholesterol acyltransferase (LCAT) deficiency (FLD) is characterized by low HDL, accumulation of an abnormal cholesterol-rich multilamellar particle called lipoprotein-X (LpX) in plasma, and renal disease. The aim of our study was to determine if LpX is nephrotoxic and to gain insight into the pathogenesis of FLD renal disease. We administered a synthetic LpX, nearly identical to endogenous LpX in its physical, chemical and biologic characteristics, to wild-type and Lcat-/- mice. Our in vitro and in vivo studies demonstrated an apoA-I and LCAT-dependent pathway for LpX conversion to HDL-like particles, which likely mediates normal plasma clearance of LpX. Plasma clearance of exogenous LpX was markedly delayed in Lcat-/- mice, which have low HDL, but only minimal amounts of endogenous LpX and do not spontaneously develop renal disease. Chronically administered exogenous LpX deposited in all renal glomerular cellular and matrical compartments of Lcat-/- mice, and induced proteinuria and nephrotoxic gene changes, as well as all of the hallmarks of FLD renal disease as assessed by histological, TEM, and SEM analyses. Extensive in vivo EM studies revealed LpX uptake by macropinocytosis into mouse glomerular endothelial cells, podocytes, and mesangial cells and delivery to lysosomes where it was degraded. Endocytosed LpX appeared to be degraded by both human podocyte and mesangial cell lysosomal PLA2 and induced podocyte secretion of pro-inflammatory IL-6 in vitro and renal Cxl10 expression in Lcat-/- mice. In conclusion, LpX is a nephrotoxic particle that in the absence of Lcat induces all of the histological and functional hallmarks of FLD and hence may serve as a biomarker for monitoring recombinant LCAT therapy. In addition, our studies suggest that LpX-induced loss of endothelial barrier function and release of cytokines by renal glomerular cells likely plays a role in the initiation and progression of FLD nephrosis.

Systemic lupus erythematosus (SLE) is characterized by increased vascular risk due to premature atherosclerosis independent of traditional risk factors. We previously proposed that IFN-α plays a crucial role in premature vascular damage in SLE. IFN-α alters the balance between endothelial cell apoptosis and vascular repair mediated by endothelial progenitor cells (EPCs) and myeloid circulating angiogenic cells (CACs). In this study, we demonstrate that IFN-α promotes an antiangiogenic signature in SLE and control EPCs/CACs, characterized by transcriptional repression of IL-1α and β, IL-1R1, and vascular endothelial growth factor A, and upregulation of IL-1R antagonist and the decoy receptor IL-1R2. IL-1β promotes significant improvement in the functional capacity of lupus EPCs/CACs, therefore abrogating the deleterious effects of IFN-α. The beneficial effects from IL-1 are mediated, at least in part, by increases in EPC/CAC proliferation, by decreases in EPC/CAC apoptosis, and by preventing the skewing of CACs toward nonangiogenic pathways. IFN-α induces STAT2 and 6 phosphorylation in EPCs/CACs, and JAK inhibition abrogates the transcriptional antiangiogenic changes induced by IFN-α in these cells. Immunohistochemistry of renal biopsies from patients with lupus nephritis, but not anti-neutrophil cytoplasmic Ab-positive vasculitis, showed this pathway to be operational in vivo, with increased IL-1R antagonist, downregulation of vascular endothelial growth factor A, and glomerular and blood vessel decreased capillary density, compared with controls. Our study introduces a novel putative pathway by which type I IFNs may interfere with vascular repair in SLE through repression of IL-1–dependent pathways. This could promote atherosclerosis and loss of renal function in this disease.

Neutrophil-specific genes are abundant in PBMC microarrays from lupus patients because of the presence of low-density granulocytes (LDGs) in mononuclear cell fractions. The functionality and pathogenicity of these LDGs have not been characterized. We developed a technique to purify LDGs from lupus PBMCs and assessed their phenotype, function, and potential role in disease pathogenesis. LDGs, their autologous lupus neutrophils, and healthy control neutrophils were compared with regard to their microbicidal and phagocytic capacities, generation of reactive oxygen species, activation status, inflammatory cytokine profile, and type I IFN expression and signatures. The capacity of LDGs to kill endothelial cells and their antiangiogenic potential were also assessed. LDGs display an activated phenotype, secrete increased levels of type I IFNs, TNF-α, and IFN-γ, but show impaired phagocytic potential. LDGs induce significant endothelial cell cytotoxicity and synthesize sufficient levels of type I IFNs to disrupt the capacity of endothelial progenitor cells to differentiate into mature endothelial cells. LDG depletion restores the functional capacity of endothelial progenitor cells. We conclude that lupus LDGs are proinflammatory and display pathogenic features, including the capacity to synthesize type I IFNs. They may play an important dual role in premature cardiovascular disease development in systemic lupus erythematosus by simultaneously mediating enhanced vascular damage and inhibiting vascular repair.

Fibroblast-like synoviocytes (FLS) and T cells can activate each other in vitro, and in vivo interactions between these cells may be important in rheumatoid arthritis (RA), yet FLS lack significant expression of CD28 ligands. We sought to identify molecules homologous to CD28 ligands that are strongly expressed by FLS, and documented strong B7-H3 expression on FLS and by fibroblasts of other tissues, which was unaffected by a variety of cytokines. Western blot analysis of FLS lysates showed predominant expression of the larger, four Ig-like domain isoform of B7-H3. Immunohistological sections of RA synovial tissue showed strong staining for B7-H3 on FLS. Cells expressing B7-H3 were distinct from but in close proximity to cells that expressed CD45, CD20, and CD3. Confocal microscopy of FLS and T cell cocultures showed localization of B7-H3 in the region of the T cell-FLS contact point, but distinct from the localization of T cell CD11a/CD18 (LFA-1) and FLS CD54 (ICAM-1). Reduction of B7-H3 expression on FLS by RNA interference affected interactions of FLS with resting T cells or cytokine-activated T cells. Resting T cells showed increased production of TNF-α, IFN-γ, and IL-2, whereas cytokine-activated T cells showed reduced cytokine production relative to control. However, cytokine production by T cells activated through their TCR was not notably altered by knock down of B7-H3. These observations suggest that B7-H3 may be important for the interactions between FLS and T cells in RA, as well as other diseases, and the outcome of such interactions depends on the activation state of the T cell.

BACKGROUND: Acquired severe HDL deficiency is relatively uncommon. It may occur with use of high doses of anabolic steroids or in severe liver diseases, which can lead to low LCAT activity and decreased apoA-I production. “Disappearing HDL Syndrome”*, a term first used by Goldberg and Mendez, refers to cases of severe HDL deficiency in patients that are not critically ill, sometimes long before the clinical or biochemical features of the underlying primary disease become evident. Disappearing HDL Syndrome can also result from an idiosyncratic reaction to medications, such as peroxisome proliferation-activated receptor (PPAR) agonists. Additionally, autoantibodies against LCAT in non-Hodgkin lymphoma have been described as a possible cause. Low high-density lipoprotein-cholesterol (HDL-C) is a risk factor for coronary artery disease. OBJECTIVE: To determine the cause of low HDL-C in patients with severe acquired HDL deficiency noted as an incidental finding associated with lymphoma and autoimmune lymphoproliferative syndrome (ALPS)** as well as following recombinant human IL10 therapy in psoriatric arthritis patients. Investigating mechanisms underlying acquired severe HDL deficiency in non-critically ill patients (“Disappearing HDL Syndrome”) could provide new insights into HDL metabolism and its role in lymphomagenesis. PATIENTS AND RESULTS: Patients with intravascular large B-cell lymphoma (IVLBCL, n=2), diffuse large B-cell lymphoma (DLBCL, n=1), and ALPS-FAS (n=1) presenting with markedly decreased HDL-C, low LDL-C and elevated triglycerides were identified. The abnormal lipoprotein profile returned to normal following therapy in all four cases (Figure). All of them were found to have markedly elevated serum interleukin-10 (IL-10) levels at presentation that also normalized following therapy. Besides accumulation of abnormal lymphocytes in lymph nodes and spleen, ALPS patients have elevated serum IL-10. In a cohort of ALPS patients with genetic mutations in FAS (n=93), IL-10 showed a strong inverse correlation with HDL-C (R2=0á3720, P CONCLUSION: Increased IL-10 causes severe HDL-C deficiency, low LDL-C and elevated triglycerides. IL-10 is thus a potent modulator of lipoprotein levels, a potential new biomarker for B-cell disorders, and a novel cause of Disappearing HDL Syndrome. Elevated IL-10 plays a key role in linking inflammation and lipoprotein metabolism. As evidence for its causality, rhIL-10 treatment in a clinical trial of psoriasitic arthritis patients precipitously lowered HDL-C. Finding elevated IL-10 as a cause of “Disappearing HDL Syndrome” in patients with 3 different types of B-cell disorders suggests that IL-10 and low HDL-C could be useful biomarkers of disease activity in these conditions. Finally, future research focusing on strategies to alter lipoprotein levels by modulating circulating IL-10 levels or its signaling pathways may be useful for developing novel targeted therapies. *Ref: Severe acquired (secondary) high-density lipoprotein deficiency. Goldberg RB, Mendez AJ. J Clin Lipidol. 2007;1:41-56 **Ref: Natural history of autoimmune lymphoproliferative syndrome associated with FAS gene mutations. Price S, Shaw PA, Seitz A et al. Blood. 2014 Mar 27;123(13):1989-99. Figure. Concomitant drop in IL-10 associated with HDL-C recovery during treatment in patients with B-cell disorders. IL-10 and HDL-C time course before and after chemotherapy in: (A, B) 2 IVLBCL and (C) 1 DLBCL patients. The first time point (“Day 0”) represents the last lipid time point before initiating chemotherapy (arrow). D. Recovery of HDL-C and IL-10 in an ALPS patient after treatment with sirolimus over a period of 6 years. P* denotes high dose (15 mg/day) prednisone pulse therapy in this 2 year old ALPS patient. Figure. Concomitant drop in IL-10 associated with HDL-C recovery during treatment in patients with B-cell disorders. IL-10 and HDL-C time course before and after chemotherapy in: (A, B) 2 IVLBCL and (C) 1 DLBCL patients. The first time point (“Day 0”) represents the last lipid time point before initiating chemotherapy (arrow). D. Recovery of HDL-C and IL-10 in an ALPS patient after treatment with sirolimus over a period of 6 years. P* denotes high dose (15 mg/day) prednisone pulse therapy in this 2 year old ALPS patient. Disclosures No relevant conflicts of interest to declare.

Introduction There is a growing body of evidence implicating aberrant dendritic cell function as a crucial component in the immunopathogenesis of systemic lupus erythematosus. The purpose of the present study was to characterize the phagocytic capacity and expression of receptors involved in pathogen recognition and self-nonself discrimination on myeloid dendritic cells from patients with lupus. Methods Unstimulated or stimulated monocyte-derived dendritic cells were obtained from lupus patients and healthy control individuals, and expression of C-type lectin receptors (mannose receptor and dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin), complement-receptor 3 and Fcγ receptors was determined by flow cytometry. Dextran uptake by lupus and control dendritic cells was also assessed by flow cytometry. Serum IFNγ was quantified by ELISA, and uptake of microbial products was measured using fluorescently labeled zymosan. Results When compared with dendritic cells from healthy control individuals, unstimulated and stimulated lupus dendritic cells displayed significantly decreased dextran uptake and mannose receptor and dendritic cell-specific intercellular adhesion molecule-grabbing nonintegrin expression. Decreased expression of the mannose receptor was associated with high serum IFNγ levels, but not with maturation status or medications. Diminished dextran uptake and mannose receptor expression correlated with lupus disease activity. There were no differences between control and lupus dendritic cells in the expression of other pattern recognition receptors or in the capacity to uptake zymosan particles Conclusions Lupus dendritic cells have diminished endocytic capacity, which correlates with decreased mannose receptor expression. While this phenomenon appears primarily intrinsic to dendritic cells, modulation by serum factors such as IFNγ could play a role. These abnormalities may be relevant to the aberrant immune homeostasis and the increased susceptibility to infections described in lupus.