Karin A. Zemski Berry

MALDI imaging of lipid biochemistry in tissues by mass spectrometry.

As a result of recent advances, remarkable images revealing the distribution of complex lipids in tissues are now generated by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Lipids are amphipathic biomolecules with hydrophobic structural characteristics made by either an initial anion thioester condensation reaction (fatty acid synthase) or by carbocation condensation of branched chain pyrophosphate intermediates (isoprene pathway).1 Lipids play essential roles in cellular function including the self-assembly of phospholipids to form the constitutive outer and inner membrane bilayer of every living cell. Specific components of these membrane phospholipids include species that contain esterified arachidonate that can be enzymatically released to a free acid and transformed to potent signaling molecules (prostaglandins, leukotrienes) with myriad biological effects. The lipid cholesterol is an essential component of bilayer membranes that has a complicated, yet highly regulated biosynthesis. Elevation of cholesterol levels (predominantly as cholesteryl esters) in blood has been implicated in heart disease and is commonly monitored in consideration of human health. Some lipid molecules play the central role in biochemical energy storage in the form of triacylglycerol molecules stored in lipid bodies within most all cells. Mass spectrometry has historically been a tool of choice in biochemical studies of lipids. The sensitivity and specificity of mass spectral data are useful to sort out the complexity of lipid structures to begin to follow biological changes. While techniques such as fluorescence confocal microscopy or ability to engineer proteins that can be expressed in cells with fluorescent tags have become the mainstream of modern biochemical research, such techniques are not amenable to most lipids due to the relatively small size of the lipid molecules and the dynamic nature of their structure in the cell. Recent developments in MALDI IMS have merged specificity of lipid identification with two-dimensional molecular mapping to enable biochemical studies of lipids across regions of a biological tissue. Several significant reasons for the success of MALDI IMS applied to lipid imaging have emerged. The first is the high abundance of various lipids in biological tissues because these hydrophobic molecules constitute the external and internal defining membranes of each cell. These membranes are almost exclusively bilayers composed of phospholipids, sphingolipids, and cholesterol that are closely packed in high local concentrations to render the membrane only semipermeable to water. A second reason is that many lipids, e.g. phospholipids, are already ionized as either phosphate anions or nitrogen centered cations and generate abundant positive or negative ions during the MALDI process. An equally important factor in the success of MALDI IMS of lipids is that the molecular weight of these biomolecules is generally below 1,000 Da, which is an optimal mass range for the most sensitive operation of modern mass spectrometers. Additionally this low molecular weight facilitates diffusion of lipids into a matrix crystal driven by the high concentration of the lipid within the microstructure of the tissue. Because of these fundamental factors coupled with the exciting potential of MALDI IMS, lipid molecules have been frequently used as substrates for the advancement of IMS methodology and instrumentation. Research groups that utilize secondary ion mass spectrometry (SIMS) imaging have embraced lipid biochemistry by moving from inorganic to biological applications, development of larger particle size beams and demonstrations of sub-micron lateral resolution.2,3 Similar development and implementation of instrumentation for MALDI IMS has leveraged lipid diversity, abundance and contrast in rodent brain samples to achieve advancements in technology.4-8 The development of different matrices useful for MALDI IMS,9-16 different methods of matrix application17-23 and different matrix modifiers24,25 have been employed in MALDI IMS experiments to establish the value and parameters of these method modifications for lipid analysis. Advances in biology have been a direct result of our ability to observe biochemical events at the micron and submicron regimes within a tissue. Having a sensitive technique that reveals molecular structure information about specific lipids in a tissue with 10-50 μm resolution and provides information relative to concentration of that lipid, has already provided insight into lipid biochemistry at the tissue level. Since lipids are products of complex, intertwined enzymatic processes, MALDI IMS data reveals the integrated solution to complex reaction pathways that define the living cell in terms of lipid biochemistry. It has become apparent to a host of scientists converging into the use of MALDI IMS from fields as diverse as neuroscience, chemistry, and instrument development that there is a richness and complexity of lipid biochemistry suggested by the exquisite, molecule specific MALDI images created in the course of developing this technology. Many reviews have focused on the technological developments of MALDI IMS of lipids with respect to the issues mentioned above.2,26,27 This review focuses on the lipid biochemistry revealed by MALDI IMS.

Dual 12/15- and 5-Lipoxygenase Deficiency in Macrophages Alters Arachidonic Acid Metabolism and Attenuates Peritonitis and Atherosclerosis in ApoE Knock-out Mice

Lipoxygenase (LO) enzymes catalyze the conversion of arachidonic acid (AA) into biologically active lipid mediators. Two members, 12/15-LO and 5-LO, regulate inflammatory responses and have been studied for their roles in atherogenesis. Both 12/15-LO and 5-LO inhibitors have been suggested as potential therapy to limit the development of atherosclerotic lesions. Here we used a genetic strategy to disrupt both 12/15-LO and 5-LO on an apolipoprotein E (apoE) atherosclerosis-susceptible background to study the impact of dual LO blockade in atherosclerosis and inflammation. Resident peritoneal macrophages are the major cell type that expresses both LO enzymes, and we verified their absence in dual LO-deficient mice. Examination of AA conversion by phorbol myristate acetate-primed and A23187-challenged macrophages from dual LO-deficient mice revealed extensive accumulation of AA with virtually no diversion into the most common cyclooxygenase (COX) products measured (prostaglandin E2 and thromboxane B2). Instead the COX-1 by-products 11-hydroxy-eicosatetraenoic acid (HETE) and 15-HETE were elevated. The interrelationship between the two LO pathways in combination with COX-1 inhibition (SC-560) also revealed striking patterns of unique substrate utilization. 5-LO- and dual LO-deficient mice exhibited an attenuated response to zymosan-induced peritoneal inflammation, emphasizing roles for 5-LO in regulating vascular permeability. We observed gender-specific attenuation of atheroma formation at 6 months of age at both the aortic root and throughout the entire aorta in chow-fed female dual LO-deficient mice. We propose that some of the inconsistent data obtained with single LO-deficient mice could be attributable to macrophage-specific patterns of altered AA metabolism.

NADPH Oxidase-dependent Generation of Lysophosphatidylserine Enhances Clearance of Activated and Dying Neutrophils via G2A

Exofacial phosphatidylserine (PS) is an important ligand mediating apoptotic cell clearance by phagocytes. Oxidation of PS fatty acyl groups (oxPS) during apoptosis reportedly mediates recognition through scavenger receptors. Given the oxidative capacity of the neutrophil NADPH oxidase, we sought to identify oxPS signaling species in stimulated neutrophils. Using mass spectrometry analysis, only trace amounts of previously characterized oxPS species were found. Conversely, 18:1 and 18:0 lysophosphatidylserine (lyso-PS), known bioactive signaling phospholipids, were identified as abundant modified PS species following activation of the neutrophil oxidase. NADPH oxidase inhibitors blocked the production of lyso-PS in vitro, and accordingly, its generation in vivo by activated, murine neutrophils during zymosan-induced peritonitis was absent in mice lacking a functional NADPH oxidase (gp91phox-/-). Treatment of macrophages with lyso-PS enhanced the uptake of apoptotic cells in vitro, an effect that was dependent on signaling via the macrophage G2A receptor. Similarly, endogenously produced lyso-PS also enhanced the G2A-mediated uptake of activated PS-exposing (but non-apoptotic) neutrophils, raising the possibility of non-apoptotic mechanisms for removal of inflammatory cells during resolution. Finally, antibody blockade of G2A signaling in vivo prolonged zymosan-induced neutrophilia in wild-type mice, whereas having no effect in gp91phox-/- mice where lyso-PS are not generated. Taken together, we show that lyso-PS are modified PS species generated following activation of the NADPH oxidase and lyso-PS signaling through the macrophage G2A functions to enhance existing receptor/ligand systems for optimal resolution of neutrophilic inflammation.

MALDI imaging MS of phospholipids in the mouse lung.

Lipid mediators are important in lung biochemistry and are derived from the enzymatic oxidation of arachidonic and docosahexaenoic acids, which are PUFAs that are present in phospholipids in cell membranes. In this study, MALDI imaging MS was used to determine the localization of arachidonate- and docosahexaenoate-containing phospholipids in mouse lung. These PUFA-containing phospholipids were determined to be uniquely abundant at the lining of small and large airways, which were unequivocally identified by immunohistochemistry. In addition, it was found that the blood vessels present in the lung were characterized by sphingomyelin molecular species, and lung surfactant phospholipids appeared evenly distributed throughout the lung parenchyma, indicating alveolar localization. This technique revealed unexpected high concentrations of arachidonate- and docosahexaenoate-containing phospholipids lining the airways in pulmonary tissue, which could serve as precursors of lipid mediators affecting airways biology.

Signaling via macrophage G2A enhances efferocytosis of dying neutrophils by augmentation of Rac activity

Phosphatidylserine (PS) and oxidized PS species have been identified as key ligands on apoptotic cells important for their recognition and removal (efferocytosis) by phagocytes, a requisite step for resolution of inflammation. We have recently demonstrated that lysophosphatidylserine (lyso-PS) generated and retained on neutrophils following short term activation of the NADPH oxidase in vitro and in vivo enhanced their clearance via signaling through the macrophage G-protein-coupled receptor G2A. Here, we investigated the signaling pathway downstream of G2A. Lyso-PS, either made endogenously in apoptosing neutrophils or supplied exogenously in liposomes along with lyso-PSneg apoptotic cells, signaled to macrophages in a G2A-dependent manner for their enhanced production of prostaglandin E2 (PGE2) via a calcium-dependent cytosolic phospholipase A2/cyclooxygenase-mediated mechanism. Subsequent signaling by PGE2 via EP2 receptors activated macrophage adenylyl cyclase and protein kinase A. These events, in turn, culminated in enhanced activity of Rac1, resulting in an increase in both the numbers of macrophages efferocytosing apoptotic cells and the numbers of cells ingested per macrophage. These data were surprising in light of previous reports demonstrating that signaling by PGE2 and adenylyl cyclase activation are associated with macrophage deactivation and inhibition of apoptotic cell uptake. Further investigation revealed that the impact of this pathway, either the enhancement or inhibition of efferocytosis, was exquisitely sensitive to concentration effects of these intermediaries. Together, these data support the hypothesis that lyso-PS presented on the surface of activated and dying neutrophils provides a tightly controlled, proresolution signal for high capacity clearance of neutrophils in acute inflammation.

MALDI Imaging of Lipids after Matrix Sublimation/Deposition

Mass spectrometric techniques have been developed to record mass spectra of biomolecules including lipids as they naturally exist within tissues and thereby permit the generation of images displaying the distribution of specific lipids in tissues, organs, and intact animals. These techniques are based on matrix-assisted laser desorption/ionization (MALDI) that requires matrix application onto the tissue surface prior to analysis. One technique of application that has recently shown significant advantages for lipid analysis is sublimation of matrix followed by vapor deposition directly onto the tissue. Explanations for enhanced sensitivity realized by sublimation/deposition related to sample temperature after a laser pulse and matrix crystal size are presented. Specific examples of sublimation/deposition in lipid imaging of various organs including brain, ocular tissue, and kidney are presented.

Spatial organization of lipids in the human retina and optic nerve by MALDI imaging mass spectrometry

MALDI imaging mass spectrometry (IMS) was used to characterize lipid species within sections of human eyes. Common phospholipids that are abundant in most tissues were not highly localized and observed throughout the accessory tissue, optic nerve, and retina. Triacylglycerols were highly localized in accessory tissue, whereas sulfatide and plasmalogen glycerophosphoethanolamine (PE) lipids with a monounsaturated fatty acid were found enriched in the optic nerve. Additionally, several lipids were associated solely with the inner retina, photoreceptors, or retinal pigment epithelium (RPE); a plasmalogen PE lipid containing DHA (22:6), PE(P-18:0/22:6), was present exclusively in the inner retina, and DHA-containing glycerophosphatidylcholine (PC) and PE lipids were found solely in photoreceptors. PC lipids containing very long chain (VLC)-PUFAs were detected in photoreceptors despite their low abundance in the retina. Ceramide lipids and the bis-retinoid, N-retinylidene-N-retinylethanolamine, was tentatively identified and found only in the RPE. This MALDI IMS study readily revealed the location of many lipids that have been associated with degenerative retinal diseases. Complex lipid localization within retinal tissue provides a global view of lipid organization and initial evidence for specific functions in localized regions, offering opportunities to assess their significance in retinal diseases, such as macular degeneration, where lipids have been implicated in the disease process.

Cytochrome P-450 4F18 Is the Leukotriene B4 ω-1/ω-2 Hydroxylase in Mouse Polymorphonuclear Leukocytes IDENTIFICATION AS THE FUNCTIONAL ORTHOLOGUE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE CYP4F3A IN THE DOWN-REGULATION OF RESPONSES TO LTB4

Leukotriene B4 (LTB4) is a potent chemoattractant for polymorphonuclear leukocytes (PMN) and other cells. Human PMN inactivate LTB4 by ω-oxidation catalyzed by cytochrome P-450 (CYP) 4F3A. The contribution of the enzymatic inactivation of LTB4 by CYP4Fs to down-regulating functional responses of cells to LTB4 is unknown. To elucidate the role of CYP4F-mediated inactivation of LTB4 in terminating the responses of PMN to LTB4 and to identify a target for future genetic studies in mice, we have identified the enzyme that catalyzes the ω-1 and ω-2 oxidation of LTB4 in mouse myeloid cells as CYP4F18. As determined by mass spectrometry, this enzyme catalyzes the conversion of LTB4 to 19-OH LTB4 and to a lesser extent 18-OH LTB4. Inhibition of CYP4F18 resulted in a marked increase in calcium flux and a 220% increase in the chemotactic response of mouse PMN to LTB4. CYP4F18 expression was induced in bone marrow-derived dendritic cells by bacterial lipopolysaccharide, a ligand for TLR4, and by poly(I·C), a ligand for TLR3. However, when bone marrow-derived myeloid dendritic cells trafficked to popliteal lymph nodes from paw pads, the expression of CYP4F18 was down-regulated. The results identify CYP4F18 as a critical protein in the regulation of LTB4 metabolism and functional responses in mouse PMN and identify it as the functional orthologue of human PMN CYP4F3A.

Phosphatidylglycerol provides short-term prophylaxis against respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) causes respiratory tract infections in young children, and significant morbidity and mortality in the elderly, immunosuppressed, and immunocompromised patients and in patients with chronic lung diseases. Recently, we reported that the pulmonary surfactant phospholipid palmitoyl-oleoyl-phosphatidylglycerol (POPG) inhibited RSV infection in vitro and in vivo by blocking viral attachment to epithelial cells. Simultaneous application of POPG along with an RSV challenge to mice markedly attenuated infection and associated inflammatory responses. Based on these findings, we expanded our studies to determine whether POPG is effective for prophylaxis and postinfection treatment for RSV infection. In vitro application of POPG at concentrations of 0.2–1.0 mg/ml at 24 h after RSV infection of HEp-2 cells suppressed interleukin-8 production up to 80% and reduced viral plaque formation by 2–6 log units. In vivo, the turnover of POPG in mice is relatively rapid, making postinfection application impractical. Intranasal administration of POPG (0.8–3.0 mg), 45 min before RSV inoculation in mice reduced viral infection by 1 log unit, suppressed inflammatory cell appearance in the lung, and suppressed virus-elicited interferon-γ production. These findings demonstrate that POPG is effective for short-term protection of mice against subsequent RSV infection and that it has potential for application in humans.

Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands

The short palate, lung and nasal epithelial clone 1 (SPLUNC1) protein is a member of the palate, lung, and nasal epithelium clone (PLUNC) family, also known as bactericidal/permeability‐increasing (BPI) fold‐containing protein, family A, member 1 (BPIFA1). SPLUNC1 is an abundant protein in human airways, but its function remains poorly understood. The lipid ligands of SPLUNC1 as well as other PLUNC family members are largely unknown, although some reports provide evidence that lipopolysaccharide (LPS) could be a lipid ligand. Unlike previous hypotheses, we found significant structural differences between SPLUNC1 and BPI. Recombinant SPLUNC1 produced in HEK 293 cells harbored several molecular species of sphingomyelin and phosphatidylcholine as its ligands. Significantly, in vitro lipid‐binding studies failed to demonstrate interactions between SPLUNC1 and LPS, lipoteichoic acid, or polymyxin B. Instead, one of the major and most important pulmonary surfactant phospholipids, dipalmitoylphosphatidylcholine (DPPC), bound to SPLUNC1 with high affinity and specificity. We found that SPLUNC1 could be the first protein receptor for DPPC. These discoveries provide insight into the specific determinants governing the interaction between SPLUNC1 and lipids and also shed light on novel functions that SPLUNC1 and other PLUNC family members perform in host defense.—Ning, F., Wang, C., Berry, K. Z., Kandasamy, P., Liu, H., Murphy, R. C., Voelker, D. R., Nho, C. W., Pan, C.‐H., Dai, S., Niu, L., Chu, H.‐W., Zhang, G. Structural characterization of the pulmonary innate immune protein SPLUNC1 and identification of lipid ligands. FASEB J. 28, 5349–5360 (2014). www.fasebj.org