This article is available online at http://www.jlr.org usually determines isotope enrichment by measuring the derivatized forms of D0 and trideuterated leucine (D3-Leu) ( 2, 3 ), a method with high cost and low sensitivity and specifi city. Recently, proteomics-based triple quadrupole multiple reaction monitoring (MRM) permitted a more practical and highly specifi c multipeptide approach to in vivo kinetic studies ( 4, 5 ). However, MRM relies on low-resolution readouts (unit mass resolution) that do not readily permit precise quantifi cation of tracer enrichment that is lower than 1%, which is common in apolipoprotein kinetics ( 5, 6 ). Factors contributing to low precision include interference by not only the sister isotope 13C15N M3 ion but also background ions. In this study, we aim to extend further the scope of in vivo kinetics by exploiting the recently developed highresolution/accurate mass parallel reaction monitoring (HR/AM-PRM) method performed on the quadrupole Orbitrap mass spectrometer ( 7,8 ). The HR/AM fragment ion scan feature has the potential to measure D3-Leu enrichment between 0.03% and 1.0%, a low incorporation range that is a consequence of a bolus-administered tracer, useful in revealing tracer-tracee relationships. (Nagoya, Japan; M.A.) and the National Institutes of Health [ R01HL107550 (M.A.); UL1 RR 025758-01 ; and R01HL095964 (F.M.S.)]. Abstract Endogenous labeling with stable isotopes is used

Interleukin-21 (IL-21) has broad actions on T- and B-cells, but its actions in innate immunity are poorly understood. Here we show that IL-21 induced apoptosis of conventional dendritic cells (cDCs) via STAT3 and Bim, and this was inhibited by granulocyte-macrophage colony-stimulating factor (GM-CSF). ChIP-Seq analysis revealed genome-wide binding competition between GM-CSF-induced STAT5 and IL-21-induced STAT3. Expression of IL-21 in vivo decreased cDC numbers, and this was prevented by GM-CSF. Moreover, repetitive α-galactosylceramide injection of mice induced IL-21 but decreased GM-CSF production by natural killer T (NKT) cells, correlating with decreased cDC numbers. Furthermore, adoptive-transfer of wild-type CD4+ T cells caused more severe colitis with increased DCs and interferon (IFN)-γ producing CD4+ T cells in Il21r−/−Rag2−/− mice (which lack T cells and have IL-21-unresponsive DCs) than in Rag2−/− mice. Thus, IL-21 and GM-CSF exhibit cross-regulatory actions on gene regulation and apoptosis, regulating cDC numbers and thereby the magnitude of the immune response.

IL-21 is a type I cytokine essential for immune cell differentiation and function. Although IL-21 can activate several STAT family transcription factors, previous studies focused mainly on the role of STAT3 in IL-21 signaling. Here, we investigated the role of STAT1 and show that STAT1 and STAT3 have at least partially opposing roles in IL-21 signaling in CD4 + T cells. IL-21 induced STAT1 phosphorylation, and this was augmented in Stat3-deficient CD4 + T cells. RNA-Seq analysis of CD4 + T cells from Stat1-and Stat3-deficient mice revealed that both STAT1 and STAT3 are critical for IL-21-mediated gene regulation. Expression of some genes, including Tbx21 and Ifng, was differentially regulated by STAT1 and STAT3. Moreover, opposing actions of STAT1 and STAT3 on IFN-γ expression in CD4 + T cells were demonstrated in vivo during chronic lymphocytic choriomeningitis infection. Finally, IL-21-mediated induction of STAT1 phosphorylation, as well as IFNG and TBX21 expression, were higher in CD4 + T cells from patients with autosomal dominant hyperIgE syndrome, which is caused by STAT3 deficiency, as well as in cells from STAT1 gain-of-function patients. These data indicate an interplay between STAT1 and STAT3 in fine-tuning IL-21 actions.) is a type I cytokine that signals via a receptor composed of IL-21R and the common cytokine receptor γ-chain, γ c (1). γ c is also shared by the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is mutated in humans with X-linked severe combined immunodeficiency (XSCID), a disease characterized by the absence of T and natural killer (NK) cells and the presence of nonfunctional B cells (2). IL-21 is primarily produced by CD4 + T cells and natural killer T (NKT) cells, but it has pleiotropic actions on both adaptive and innate immune cells, including T, B, NK, NKT, and dendritic cells (1). In T cells, IL-21 can act as a comitogen and cooperates with IL-7 and IL-15 to expand CD8 + T cells (3), promotes Th17 differentiation (4-6), and induces BCL6 expression (7) to promote T follicular helper cell development (8, 9). In B cells, IL-21 promotes plasma cell differentiation (10, 11), and in combination with IL-4, drives IgG1 and IgG3 class switch (11,12). Defective signaling by IL-21 appears to substantially explain the B-cell defect observed in patients with XSCID (11, 13). Furthermore, IL-21 can enhance the cytotoxic activity of NK and NKT cells (1) and induce the apoptosis of conventional dendritic cells (14).IL-21 activates multiple signaling pathways, including the JAK-STAT, PI 3-kinase (PI3K), and MAPK pathways (15). Of these, the JAK-STAT pathway has been most extensively studied. IL-21 induces phosphorylation of JAK1 and JAK3, which in turn leads to phosphorylation and nuclear translocation of STAT3, which then binds to IFN-γ-activated sequence (GAS) motifs and modulates expression of IL-21-responsive genes. IL-21 also activates STAT1, but the function of IL-21-activated STAT1 is largely unknown, although IL-21 was suggested to use STAT1 to promote CD8 + T-cell cytotoxicity and ap...

Objective: HDL in plasma is a heterogeneous group of lipoproteins typically containing apoA-I as the principal protein. Most HDLs contain additional proteins from a palate of nearly 100 HDL-associated polypeptides. We hypothesized that some of these proteins define distinct and stable apoA-I HDL subspecies with unique proteomes that drive function and associations with disease. Approach and Results: We produced 17 plasma pools from 80 normolipidemic human participants (32 male, 48 female; aged 21 to 66 years). Using immunoaffinity isolation techniques, we isolated apoA-I containing species from plasma and then used antibodies to 16 additional HDL protein components to isolate compositional subspecies. We characterized previously described HDL subspecies containing apoA-II, apoC-III and apoE; and 13 novel HDL subspecies defined by presence of apoA-IV, apoC-I, apoC-II, apoJ, alpha-1-antitrypsin, alpha-2-macroglobulin, plasminogen, fibrinogen, ceruloplasmin, haptoglobin, paraoxonase-1, apoL-I, or complement C3. The novel species ranged in abundance from 1–18% of total plasma apoA-I. Their concentrations were stable over time as demonstrated by intra-class correlations in repeated sampling from the same participants over 3–24 months (0.33 – 0.86; mean 0.62). Some proteomes of the subspecies relative to total HDL were strongly correlated, often among subspecies defined by similar functions: lipid metabolism, hemostasis, anti-oxidant, or anti-inflammatory. Permutation analysis showed that the proteomes of 12 of the 16 subspecies differed significantly from that of total HDL. Conclusions: Taken together, correlation and permutation analyses support speciation of HDL. Functional studies of these novel subspecies and determination of their relation to diseases may provide new avenues to understand the HDL system of lipoproteins.

Objective: Clinical evidence has linked low HDL (high-density lipoprotein) cholesterol levels with high cardiovascular disease risk; however, its significance as a therapeutic target remains unestablished. We hypothesize that HDLs functional heterogeneity is comprised of metabolically distinct proteins, each on distinct HDL sizes and that are affected by diet. Approach and Results: Twelve participants were placed on 2 healthful diets high in monounsaturated fat or carbohydrate. After 4 weeks on each diet, participants completed a metabolic tracer study. HDL was isolated by Apo (apolipoprotein) A1 immunopurification and separated into 5 sizes. Tracer enrichment and metabolic rates for 8 HDL proteins—ApoA1, ApoA2, ApoC3, ApoE, ApoJ, ApoL1, ApoM, and LCAT (lecithin-cholesterol acyltransferase)—were determined by parallel reaction monitoring and compartmental modeling, respectively. Each protein had a unique, size-specific distribution that was not altered by diet. However, carbohydrate, when replacing fat, increased the fractional catabolic rate of ApoA1 and ApoA2 on alpha3 HDL; ApoE on alpha3 and alpha1 HDL; and ApoM on alpha2 HDL. Additionally, carbohydrate increased the production of ApoC3 on alpha3 HDL and ApoJ and ApoL1 on the largest alpha0 HDL. LCAT was the only protein studied that diet did not affect. Finally, global proteomics showed that diet did not alter the distribution of the HDL proteome across HDL sizes. Conclusions: This study demonstrates that HDL in humans is composed of a complex system of proteins, each with its own unique size distribution, metabolism, and diet regulation. The carbohydrate-induced hypercatabolic state of HDL proteins may represent mechanisms by which carbohydrate alters the cardioprotective properties of HDL.

HDLs are a family of heterogeneous particles that vary in size, composition, and function. The structure of most HDLs is maintained by two scaffold proteins, apoA-I and apoA-II, but up to 95 other "accessory" proteins have been found associated with the particles. Recent evidence suggests that these accessory proteins are distributed across various subspecies and drive specific biological functions. Unfortunately, our understanding of the molecular composition of such subspecies is limited. To begin to address this issue, we separated human plasma and HDL isolated by ultracentrifugation (UC-HDL) into particles with apoA-I and no apoA-II (LpA-I) and those with both apoA-I and apoA-II (LpA-I/A-II). MS studies revealed distinct differences between the subfractions. LpA-I exhibited significantly more protein diversity than LpA-I/A-II when isolated directly from plasma. However, this difference was lost in UC-HDL. Most LpA-I/A-II accessory proteins were associated with lipid transport pathways, whereas those in LpA-I were associated with inflammatory response, hemostasis, immune response, metal ion binding, and protease inhibition. We found that the presence of apoA-II enhanced ABCA1-mediated efflux compared with LpA-I particles. This effect was independent of the accessory protein signature suggesting that apoA-II induces a structural change in apoA-I in HDLs.

Recent in vivo tracer studies demonstrated that targeted mass spectrometry (MS) on the Q Exactive Orbitrap could determine the metabolism of HDL proteins 100s-fold less abundant than apolipoprotein A1 (APOA1). In this study, we demonstrate that the Orbitrap Lumos can measure tracer in proteins whose abundances are 1000s-fold less than APOA1, specifically the lipid transfer proteins phospholipid transfer protein (PLTP), cholesterol ester transfer protein (CETP), and lecithin-cholesterol acyl transferase (LCAT). Relative to the Q Exactive, the Lumos improved tracer detection by reducing tracer enrichment compression, thereby providing consistent enrichment data across multiple HDL sizes from 6 participants. We determined by compartmental modeling that PLTP is secreted in medium and large HDL (alpha2, alpha1, and alpha0) and is transferred from medium to larger sizes during circulation from where it is catabolized. CETP is secreted mainly in alpha1 and alpha2 and remains in these sizes during circulation. LCAT is secreted mainly in medium and small HDL (alpha2, alpha3, prebeta). Unlike PLTP and CETP, LCAT’s appearance on HDL is markedly delayed, indicating that LCAT may reside for a time outside of systemic circulation before attaching to HDL in plasma. The determination of these lipid transfer proteins’ unique metabolic structures was possible due to advances in MS technologies.

We developed an automated quantification workflow for PRM-enabled detection of D3-Leu labeled apoA-I in HDL isolated from humans. Subjects received a bolus injection of D3-Leu and blood was drawn at seven time points over three days. HDL was isolated and separated into six size fractions for subsequent proteolysis and PRM analysis for the detection of D3-Leu signal from ~0.03 to 0.6 % enrichment. We implemented an intensity-based quantification approach that takes advantage of high resolution/accurate mass PRM scans to identify the D3-Leu 2HM3 ion from non-specific peaks. Our workflow includes five modules for extracting the targeted PRM peak intensities (XPIs): Peak centroiding, noise removal, fragment ion matching using Δm/z windows, nine intensity quantification options, and validation and visualization outputs. We optimized the XPI workflow using in vitro synthesized and clinical samples of D0/D3-Leu labeled apoA-I. Three subjects’ apoA-I enrichment curves in six HDL size fractions, and LCAT, apoA-II and apoE from two size fractions were generated within a few hours. Our PRM strategy and automated quantification workflow will expedite the turnaround of HDL apoA-I metabolism data in clinical studies that aim to understand and treat the mechanisms behind dyslipidemia.