Hypoxia Is a Dominant Remodeler of the Effector T Cell Surface Proteome Relative to Activation and Regulatory T Cell Suppression

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However, how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here, using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture–based quantitative cell surface capture glycoproteomics, we examined how two immunosuppressive TME factors, regulatory T cells (Tregs) and hypoxia, globally affect the activated CD8⁺ surface proteome (surfaceome). Surprisingly, coculturing primary CD8⁺ T cells with Tregs only modestly affected the CD8⁺ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast, hypoxia drastically altered the CD8⁺ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4⁺ T cell surfaceome similarly responded to hypoxia, revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function.     

B cells from Patients with Rheumatoid Arthritis Show Conserved CD39-Mediated Regulatory Function and increased CD39 Expression After Positive Response to Therapy

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by progressive joint destruction associated with increased pro-inflammatory mediators. In inflammatory microenvironments, exogenous ATP (eATP) is hydrolyzed to adenosine, which exerts immunosuppressive effects, by the consecutive action of the ectonucleotidases CD39 and CD73. Mature B cells constitutively express both ectonucleotidases, converting these cells to potential suppressors. Here, we assessed CD39 and CD73 expression on B cells from treated or untreated patients with RA. Neither the frequency of CD73⁺CD39⁺ and CD73^-CD39⁺ B cell subsets nor the levels of CD73 and CD39 expression on B cells from untreated or treated RA patients showed significant changes in comparison to healthy controls (HC). CpG+IL-2-stimulated B cells from HC or untreated RA patients increased their CD39 expression, and suppressed CD4⁺ and CD8⁺ T cell proliferation and intracellular TNF-production. A CD39 inhibitor significantly restored proliferation and TNF-producing capacity in CD4⁺ T cells, but not in CD8⁺ T cells, from HC and untreated RA patients, indicating that B cells from untreated RA patients conserved CD39-mediated regulatory function. Good responder patients to therapy (R-RA) exhibited an increased CD39 but not CD73 expression on B cells after treatment, while most of the non-responder (NR) patients showed a reduction in ectoenzyme expression. The positive changes of CD39 expression on B cells exhibited a negative correlation with disease activity and rheumatoid factor levels. Our results suggest modulating the ectoenzymes/ADO pathway as a potential therapy target for improving the course of RA.     

Cholesterol metabolism: from lipidomics to immunology

Oxysterols, the oxidized forms of cholesterol or of its precursors, are formed in the first steps of cholesterol metabolism. Oxysterols have interested chemists, biologists, and physicians for many decades, but their exact biological relevance in vivo, other than as intermediates in bile acid biosynthesis, has long been debated. However, in the first quarter of this century, a role for side-chain oxysterols and their C-7 oxidized metabolites has been convincingly established in the immune system. 25-Hydroxycholesterol has been shown to be synthesized by macrophages in response to the activation of Toll-like receptors and to offer protection against microbial pathogens, whereas 7α,25-dihydroxycholesterol has been shown to act as a chemoattractant to lymphocytes expressing the G protein-coupled receptor Epstein-Barr virus-induced gene 2 and to be important in coordinating the action of B cells, T cells, and dendritic cells in secondary lymphoid tissue. There is a growing body of evidence that not only these two oxysterols but also many of their isomers are of importance to the proper function of the immune system. Here, we review recent findings related to the roles of oxysterols in immunology.     

High-density lipoproteins mediate small RNA intercellular communication between dendritic cells and macrophages

HDL are dynamic transporters of diverse molecular cargo and play critical roles in lipid metabolism and inflammation. We have previously reported that HDL transport both host and nonhost small RNAs (sRNA) based on quantitative PCR and sRNA sequencing approaches; however, these methods require RNA isolation steps which have potential biases and may not isolate certain forms of RNA molecules from samples. HDL have also been reported to accept functional sRNAs from donor macrophages and deliver them to recipient endothelial cells; however, using PCR to trace HDL-sRNA intercellular communication has major limitations. The present study aims to overcome these technical barriers and further understand the pathways involved in HDL-mediated bidirectional flux of sRNAs between immune cells. To overcome these technical limitations, SYTO RNASelect, a lipid-penetrating RNA dye, was used to quantify a) overall HDL-sRNA content, b) bidirectional flux of sRNAs between HDL and immune cells, c) HDL-mediated intercellular communication between immune cells, and d) HDL-mediated RNA export changes in disease. Live cell imaging and loss-of-function assays indicate that the endo-lysosomal system plays a critical role in macrophage storage and export of HDL-sRNAs. These results identify HDL as a substantive mediator of intercellular communication between immune cells and demonstrate the importance of endocytosis for recipient cells of HDL-sRNAs. Utilizing a lipid-penetrating RNA-specific fluorescence dye, we were able to both quantify the absolute concentration of sRNAs transported by HDL and characterize HDL-mediated intercellular RNA transport between immune cells.     

Pulmonary surfactant protein B carried by HDL predicts incident CVD in patients with type 1 diabetes

Atherosclerotic CVD is the major cause of death in patients with type 1 diabetes mellitus (T1DM). Alterations in the HDL proteome have been shown to associate with prevalent CVD in T1DM. We therefore sought to determine which proteins carried by HDL might predict incident CVD in patients with T1DM. Using targeted MS/MS, we quantified 50 proteins in HDL from 181 T1DM subjects enrolled in the prospective Coronary Artery Calcification in Type 1 Diabetes study. We used Cox proportional regression analysis and a case-cohort design to test associations of HDL proteins with incident CVD (myocardial infarction, coronary artery bypass grafting, angioplasty, or death from coronary heart disease). We found that only one HDL protein—SFTPB (pulmonary surfactant protein B)—predicted incident CVD in all the models tested. In a fully adjusted model that controlled for lipids and other risk factors, the hazard ratio was 2.17 per SD increase of SFTPB (95% confidence interval, 1.12–4.21, P = 0.022). In addition, plasma fractionation demonstrated that SFTPB is nearly entirely bound to HDL. Although previous studies have shown that high plasma levels of SFTPB associate with prevalent atherosclerosis only in smokers, we found that SFTPB predicted incident CVD in T1DM independently of smoking status and a wide range of confounding factors, including HDL-C, LDL-C, and triglyceride levels. Because SFTPB is almost entirely bound to plasma HDL, our observations support the proposal that SFTPB carried by HDL is a marker—and perhaps mediator—of CVD risk in patients with T1DM.     

Immune Checkpoint Blockade Augments Changes Within Oncolytic Virus-induced Cancer MHC-I Peptidome,Creating Novel Antitumor CD8 T Cell Reactivities

The combination cancer immunotherapies with oncolytic virus (OV) and immune checkpoint blockade (ICB) reinstate otherwise dysfunctional antitumor CD8 T cell responses. One major mechanism that aids such reinstatement of antitumor CD8 T cells involves the availability of new class I major histocompatibility complex (MHC-I)-bound tumor epitopes following therapeutic intervention. Thus, therapy-induced changes within the MHC-I peptidome hold the key to understanding the clinical implications for therapy-reinstated CD8 T cell responses. Here, using mass spectrometry–based immuno-affinity methods and tumor-bearing animals treated with OV and ICB (alone or in combination), we captured the therapy-induced alterations within the tumor MHC-I peptidome, which were then tested for their CD8 T cell response-stimulating activity. We found that the oncolytic reovirus monotherapy drives up- as well as downexpression of tumor MHC-I peptides in a cancer type and oncolysis susceptibility dependent manner. Interestingly, the combination of reovirus + ICB results in higher numbers of differentially expressed MHC-I-associated peptides (DEMHCPs) relative to either monotherapies. Most importantly, OV+ICB-driven DEMHCPs contain biologically active epitopes that stimulate interferon-gamma responses in cognate CD8 T cells, which may mediate clinically desired antitumor attack and cancer immunoediting. These findings highlight that the therapy-induced changes to the MHC-I peptidome contribute toward the reinstated antitumor CD8 T cell attack established following OV + ICB combination cancer immunotherapy.     

10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr^?/?) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.     

Human apoA-I[Lys107del] mutation affects lipid surface behavior of apoA-I and its ability to form large nascent HDL

Population studies have found that a natural human apoA-I variant, apoA-I[K107del], is strongly associated with low HDL-C but normal plasma apoA-I levels. We aimed to reveal properties of this variant that contribute to its unusual phenotype associated with atherosclerosis. Our oil-drop tensiometry studies revealed that compared to WT, recombinant apoA-I[K107del] adsorbed to surfaces of POPC-coated triolein drops at faster rates, remodeled the surfaces to a greater extent, and was ejected from the surfaces at higher surface pressures on compression of the lipid drops. These properties may drive increased binding of apoA-I[K107del] to and its better retention on large triglyceride-rich lipoproteins, thereby increasing the variant’s content on these lipoproteins. While K107del did not affect apoA-I capacity to promote ABCA1-mediated cholesterol efflux from J774 cells, it impaired the biogenesis of large nascent HDL particles resulting in the formation of predominantly smaller nascent HDL. Size-exclusion chromatography of spontaneously reconstituted 1,2-dimyristoylphosphatidylcholine-apoA-I complexes showed that apoA-I[K107del] had a hampered ability to form larger complexes but formed efficiently smaller-sized complexes. CD analysis revealed a reduced ability of apoA-I[K107del] to increase α-helical structure on binding to 1,2-dimyristoylphosphatidylcholine or in the presence of trifluoroethanol. This property may hinder the formation of large apoA-I[K107del]-containing discoidal and spherical HDL but not smaller HDL. Both factors, the increased content of apoA-I[K107del] on triglyceride-rich lipoproteins and the impaired ability of the variant to stabilize large HDL particles resulting in reduced lipid:protein ratios in HDL, may contribute to normal plasma apoA-I levels along with low HDL-C and increased risk for CVD.     

Hypertriglyceridemic hyperapoB and the development and resolution of nonalcoholic fatty liver disease: a cohort study

Hypertriglyceridemic hyperapoB is an adverse lipoprotein phenotype characterized by low high density lipoprotein (HDL) cholesterol, high triglycerides, high apolipoprotein B (ApoB), and low low density lipoprotein (LDL) cholesterol to ApoB ratio. We investigated whether and to what extent hypertriglyceridemic hyperapoB associates with the incidence and resolution of nonalcoholic fatty liver disease (NAFLD). This prospective cohort study included 9,019 Chinese participants 40 years or older, from 2010 to 2015. Logistic regression models were used to examine the odds ratios (ORs) for the incidence and resolution of NAFLD associated with the hypertriglyceridemic hyperapoB lipoprotein phenotype and individual lipid and lipoprotein parameters. During a median 4.3 years of follow-up, compared with participants with optimal phenotype, the fully adjusted ORs (95% CIs) for participants with hypertriglyceridemic hyperapoB were 2.75 (1.91, 3.95) and 0.57 (0.33, 1.00) for incidence and resolution of NAFLD, respectively. These associations were consistent across subgroup participants with varied demographic, lifestyle, and metabolic status. Individually, each unit increase in HDL cholesterol (OR: 0.98; 95% CI: 0.97, 0.99), natural logarithm-transformed triglycerides (1.89; 1.52, 2.36), and ApoB (1.006; 1.002, 1.011) was independently associated with NAFLD incidence, and only triglycerides (0.77; 0.60, 0.99) was independently associated with NAFLD resolution. Our findings suggest that Chinese adults with hypertriglyceridemic hyperapoB have a higher risk of NAFLD incidence and a lower likelihood of NAFLD resolution. These associations were stable among adults with different demographic, lifestyle, and metabolic status, supporting hypertriglyceridemic hyperapoB as a valuable clinical marker for the prevention and control of NAFLD.     

A Single-Cell Atlas of Tumor-Infiltrating Immune Cells in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with limited treatment options. To guide the design of more effective immunotherapy strategies, mass cytometry was employed to characterize the cellular composition of the PDAC-infiltrating immune cells. The expression of 33 protein markers was examined at the single-cell level in more than two million immune cells from four types of clinical samples, including PDAC tumors, normal pancreatic tissues, chronic pancreatitis tissues, and peripheral blood. Based on the analyses, we identified 23 distinct T-cell phenotypes, with some cell clusters exhibiting aberrant frequencies in the tumors. Programmed cell death protein 1 (PD-1) was extensively expressed in CD4⁺ and CD8⁺ T cells and coexpressed with both stimulatory and inhibitory immune markers. In addition, we observed elevated levels of functional markers, such as CD137L and CD69, in PDAC-infiltrating immune cells. Moreover, the combination of PD-1 and CD8 was used to stratify PDAC tumors from The Cancer Genome Atlas database into three immune subtypes, with S1 (PD-1⁺CD8⁺) exhibiting the best prognosis. Further analysis suggested distinct molecular mechanisms for immune exclusion in different subtypes. Taken together, the single-cell protein expression data depicted a detailed cell atlas of the PDAC-infiltrating immune cells and revealed clinically relevant information regarding useful cell phenotypes and targets for immunotherapy development.     

Regulation of astrocyte lipid metabolism and ApoE secretion by the microglial oxysterol, 25-hydroxycholesterol

Neuroinflammation, a major hallmark of Alzheimer’s disease and several other neurological and psychiatric disorders, is often associated with dysregulated cholesterol metabolism. Relative to homeostatic microglia, activated microglia express higher levels of Ch25h, an enzyme that hydroxylates cholesterol to produce 25-hydroxycholesterol (25HC). 25HC is an oxysterol with interesting immune roles stemming from its ability to regulate cholesterol metabolism. Since astrocytes synthesize cholesterol in the brain and transport it to other cells via ApoE-containing lipoproteins, we hypothesized that secreted 25HC from microglia may influence lipid metabolism as well as extracellular ApoE derived from astrocytes. Here, we show that astrocytes take up externally added 25HC and respond with altered lipid metabolism. Extracellular levels of ApoE lipoprotein particles increased after treatment of astrocytes with 25HC without an increase in Apoe mRNA expression. In mouse astrocytes-expressing human ApoE3 or ApoE4, 25HC promoted extracellular ApoE3 better than ApoE4. Increased extracellular ApoE was due to elevated efflux from increased Abca1 expression via LXRs as well as decreased lipoprotein reuptake from suppressed Ldlr expression via inhibition of SREBP. 25HC also suppressed expression of Srebf2, but not Srebf1, leading to reduced cholesterol synthesis in astrocytes without affecting fatty acid levels. We further show that 25HC promoted the activity of sterol-o-acyl transferase that led to a doubling of the amount of cholesteryl esters and their concomitant storage in lipid droplets. Our results demonstrate an important role for 25HC in regulating astrocyte lipid metabolism.     

Proteomics Investigation of Diverse Serological Patterns in COVID-19

Serum antibodies IgM and IgG are elevated during Coronavirus Disease 2019 (COVID-19) to defend against viral attacks. Atypical results such as negative and abnormally high antibody expression were frequently observed whereas the underlying molecular mechanisms are elusive. In our cohort of 144 COVID-19 patients, 3.5% were both IgM and IgG negative, whereas 29.2% remained only IgM negative. The remaining patients exhibited positive IgM and IgG expression, with 9.3% of them exhibiting over 20-fold higher titers of IgM than the others at their plateau. IgG titers in all of them were significantly boosted after vaccination in the second year. To investigate the underlying molecular mechanisms, we classed the patients into four groups with diverse serological patterns and analyzed their 2-year clinical indicators. Additionally, we collected 111 serum samples for TMTpro-based longitudinal proteomic profiling and characterized 1494 proteins in total. We found that the continuously negative IgM and IgG expression during COVID-19 were associated with mild inflammatory reactions and high T cell responses. Low levels of serum IgD, inferior complement 1 activation of complement cascades, and insufficient cellular immune responses might collectively lead to compensatory serological responses, causing overexpression of IgM. Serum CD163 was positively correlated with antibody titers during seroconversion. This study suggests that patients with negative serology still developed cellular immunity for viral defense and that high titers of IgM might not be favorable to COVID-19 recovery.     

Dalcetrapib and anacetrapib increase apolipoprotein E-containing HDL in rabbits and humans

The large HDL particles generated by administration of cholesteryl ester transfer protein inhibitors (CETPi) remain poorly characterized, despite their potential importance in the routing of cholesterol to the liver for excretion, which is the last step of the reverse cholesterol transport. Thus, the effects of the CETPi dalcetrapib and anacetrapib on HDL particle composition were studied in rabbits and humans. The association of rabbit HDL to the LDL receptor (LDLr) in vitro was also evaluated. New Zealand White rabbits receiving atorvastatin were treated with dalcetrapib or anacetrapib. A subset of patients from the dal-PLAQUE-2 study treated with dalcetrapib or placebo were also studied. In rabbits, dalcetrapib and anacetrapib increased HDL-C by more than 58% (P < 0.01) and in turn raised large apo E-containing HDL by 66% (P < 0.001) and 59% (P < 0.01), respectively. Additionally, HDL from CETPi-treated rabbits competed with human LDL for binding to the LDLr on HepG2 cells more than control HDL (P < 0.01). In humans, dalcetrapib increased concentrations of large HDL particles (+69%, P < 0.001) and apo B-depleted plasma apo E (+24%, P < 0.001), leading to the formation of apo E-containing HDL (+47%, P < 0.001) devoid of apo A-I. Overall, in rabbits and humans, CETPi increased large apo E-containing HDL particle concentration, which can interact with hepatic LDLr. The catabolism of these particles may depend on an adequate level of LDLr to contribute to reverse cholesterol transport.     

Low concentrations of medium-sized HDL particles predict incident CVD in chronic kidney disease patients

Patients with chronic kidney disease (CKD) are at high risk for CVD. However, traditional CVD risk factors cannot completely explain the increased risk. Altered HDL proteome is linked with incident CVD in CKD patients, but it is unclear whether other HDL metrics are associated with incident CVD in this population. In the current study, we analyzed samples from two independent prospective case-control cohorts of CKD patients, the Clinical Phenotyping and Resource Biobank Core (CPROBE) and the Chronic Renal Insufficiency Cohort (CRIC). We measured HDL particle sizes and concentrations (HDL-P) by calibrated ion mobility analysis and HDL cholesterol efflux capacity (CEC) by cAMP-stimulated J774 macrophages in 92 subjects from the CPROBE cohort (46 CVD and 46 controls) and in 91 subjects from the CRIC cohort (34 CVD and 57 controls). We tested associations of HDL metrics with incident CVD using logistic regression analysis. No significant associations were found for HDL-C or HDL-CEC in either cohort. Total HDL-P was only negatively associated with incident CVD in the CRIC cohort in unadjusted analysis. Among the six sized HDL subspecies, only medium-sized HDL-P was significantly and negatively associated with incident CVD in both cohorts after adjusting for clinical confounders and lipid risk factors with odds ratios (per 1-SD) of 0.45 (0.22–0.93, P = 0.032) and 0.42 (0.20–0.87, P = 0.019) for CPROBE and CRIC cohorts, respectively. Our observations indicate that medium-sized HDL-P—but not other-sized HDL-P or total HDL-P, HDL-C, or HDL-CEC—may be a prognostic cardiovascular risk marker in CKD.     

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1^−/− mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND-fed Cyp7b1^−/− mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1^−/− mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1^−/− mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.     

HDL Isolated by Immunoaffinity,Ultracentrifugation, or Precipitation is Compositionally and Functionally Distinct

The HDL proteome has been widely recognized as an important mediator of HDL function. While a variety of HDL isolation methods exist, their impact on the HDL proteome and its associated function remain largely unknown. Here, we compared three of the most common methods for HDL isolation, namely immunoaffinity (IA), density gradient ultracentrifugation (UC), and dextran-sulfate precipitation (DS), in terms of their effects on the HDL proteome and associated functionalities. We used state-of-the-art mass spectrometry to identify 171 proteins across all three isolation methods. IA-HDL contained higher levels of paraoxonase 1, apoB, clusterin, vitronectin, and fibronectin, while UC-HDL had higher levels of apoA2, apoC3, and α-1-antytrypsin. DS-HDL was enriched with apoA4 and complement proteins, while the apoA2 content was very low. Importantly, size-exclusion chromatography analysis showed that IA-HDL isolates contained subspecies in the size range above 12 nm, which were entirely absent in UC-HDL and DS-HDL isolates. Analysis of these subspecies indicated that they primarily consisted of apoA1, IGκC, apoC1, and clusterin. Functional analysis revealed that paraoxonase 1 activity was almost completely lost in IA-HDL, despite high paraoxonase content. We observed that the elution conditions, using 3M thiocyanate, during IA resulted in an almost complete loss of paraoxonase 1 activity. Notably, the cholesterol efflux capacity of UC-HDL and DS-HDL was significantly higher compared to IA-HDL. Together, our data clearly demonstrate that the isolation procedure has a substantial impact on the composition, subclass distribution, and functionality of HDL. In summary, our data show that the isolation procedure has a significant impact on the composition, subclass distribution and functionality of HDL. Our data can be helpful in the comparison, replication and analysis of proteomic datasets of HDL.     

Immunoregulatory effect of mesenchymal stem cell via mitochondria signaling pathways in allergic asthma

Asthma is a complicated lung disease, which has increased morbidity and mortality rates in worldwide. There is an overlap between asthma pathophysiology and mitochondrial dysfunction and MSCs may have regulatory effect on mitochondrial dysfunction and treats asthma. Therefore, immune-modulatory effect of MSCs and mitochondrial signaling pathways in asthma was studied.After culturing of MSCs and producing asthma animal model, the mice were treated with MSCs via IV via IT. BALf's eosinophil Counting, The levels of IL-4, −5, −13, −25, –33, INF-γ, Cys-LT, LTB4, LTC4, mitochondria genes expression of COX-1, COX-2, ND1, Nrf2, Cytb were measured and lung histopathological study were done.BALf's eosinophils, the levels of IL-4, −5, −13, −25, –33, LTB4, LTC4, Cys-LT, the mitochondria genes expression (COX-1, COX-2, Cytb and ND-1), perivascular and peribronchial inflammation, mucus hyper-production and hyperplasia of the goblet cell in pathological study were significantly decreased in MSCs-treated asthma mice and reverse trend was found about Nrf-2 gene expression, IFN-γ level and ratio of the INF-γ/IL-4.MSC therapy can control inflammation, immune-inflammatory factors in asthma and mitochondrial related genes, and prevent asthma immune-pathology.     

β-Carotene enhances the expression of inflammation-related genes and histone H3 K9 acetylation,K4 dimethylation,and K36 trimethylation around these genes in juvenile macrophage-like THP-1 cells

β-Carotene is converted into vitamin A in the body and can remove reactive oxygen species. However, it is still unclear whether β-carotene alters the expression levels of inflammation-related genes in macrophages and how this is regulated. In the present study, we investigated whether the administration of β-carotene under hyperglycemic conditions altered the expression level of inflammation-related genes and whether any observed differences were associated with changes in histone modifications in juvenile macrophage-like THP-1 cells. THP-1 cells (from a human monocytic leukemia cell line) were cultured in low glucose (5 mM), high glucose (25 mM), or high glucose (25 mM) + β-carotene (5 μM) media for 1 day, and mRNA expression levels of genes related to oxidative stress and inflammation, and histone modifications were determined by mRNA microarray and qRT-PCR analyses, and chromatin immunoprecipitation assays, respectively. The expression of inflammation-related genes, such as IL31RA, CD38, and NCF1B, and inflammation-associated signaling pathway genes, such as ITGAL, PRAM1, and CSF3R, were upregulated by β-carotene under high-glucose conditions. Under these conditions, histone H3 lysine 4 (K4) demethylation, H3K36 trimethylation, and H3K9 acetylation around the CD38, NCF1B, and ITGAL genes were higher in β-carotene-treated cells than in untreated cells. Treatment of juvenile macrophage-like THP-1 cells with β-carotene under these high glucose conditions induced the expression of inflammation-related genes, K9 acetylation, and K4 di- and K36 trimethylation of histone H3 around these genes.     

Quantitative characterizations of the cholesterol-related pathways in the retina and brain of hamsters

The retina and brain are separated from the systemic circulation by the anatomical barriers, which are permeable (the outer blood-retinal barrier) and impermeable (the blood-brain and inner blood-retina barriers) to cholesterol. Herein we investigated whether whole-body cholesterol maintenance affects cholesterol homeostasis in the retina and brain. We used hamsters, whose whole-body cholesterol handling is more similar to those in humans than in mice, and conducted separate administrations of deuterated water and deuterated cholesterol. We assessed the quantitative significance of the retinal and brain pathways of cholesterol input and compared the results with those from our previous studies in mice. The utility of the measurements in the plasma of deuterated 24-hydroxycholesterol, the major cholesterol elimination product from the brain, was investigated as well. We established that despite a sevenfold higher serum LDL to HDL ratio and other cholesterol-related differences, in situ biosynthesis remained the major source of cholesterol for hamster retina, although its quantitative significance was reduced to 53% as compared to 72%–78% in the mouse retina. In the brain, the principal pathway of cholesterol input was also the same, in situ biosynthesis, accounting for 94% of the total brain cholesterol input (96% in mice); the interspecies differences pertained to the absolute rates of the total cholesterol input and turnover. We documented the correlations between deuterium enrichments of the brain 24-hydroxycholesterol, brain cholesterol, and plasma 24-hydroxycholesterol, which suggested that deuterium enrichment of plasma 24-hydroxycholesteol could be an in vivo marker of cholesterol elimination and turnover in the brain.