• Establishing a Lipid Model of Host-Pathogen Interaction Using Multimodal Mass Spectrometry Imaging in a Francisella Infection

      Scott, Alison June; Ernst, Robert K.; 0000-0001-6969-4707 (2015)
      Host membranes are intimately involved in the immune response to any infection, including formation of lipid docking sites for proteins, organization of immune signaling complexes in lipid rafts, and maintaining a reservoir of fatty acids that contribute to acute inflammation. Francisella species maintain several host immune evasion strategies, one of which involves induction of the immunomodulatory lipid prostaglandin E2 (PGE2). The source of PGE2 is arachidonic acid (AA), a structural component of membrane phospholipids. Mass spectrometry imaging (MSI) was used to map and characterize both host- and pathogen-borne lipids using Francisella infected spleens in a murine model. Here, we identified and mapped the unique bacterial molecule, lipid A within infected mouse spleens by MALDI-MSI, confirming the in vivo structure (m/z 1665.1) in a mammalian infection. Francisella lipid A mapped primarily to the red pulp of the spleen, with signal first appearing between 24 and 36 hours post-infection, corresponding to the onset of bacteremia. Numerous changes in host lipid levels were correlated with progression of the infection. A phosphatidylinositol species, 1-stearoyl, 2-arachidonyl phosphatidylinositol (SAPI) was identified in the periphery of the splenic white pulp, suggesting a cell-specific origin. SAPI abundance peaks at 24 hours and is depleted in the timepoints preceding lethality (48 to 60 hours). In vitro reports demonstrate that SAPI is the earliest source of AA in activated macrophages. We have subsequently linked importation of SAPI into the spleen by monocytic infiltrates, which increases the total SAPI load. Additionally, accumulation of cholesterol was observed by SIMS-Imaging in the infected spleens and may be another indicator of immune infiltration. These data highlight a role for newly immigrant cells in contributing to the pool of total inflammatory lipids. Here, MSI is presented as a new approach to studying lipid-level host-pathogen interactions, facilitating targeted and untargeted discovery.
    • Tissue Lipid Analysis via MALDI Imaging (MALDI-IMS)

      Feldman, Steven G.; Scott, Alison June; Ernst, Robert K. (2013-04-11)
      Mammalian tissue contains a complex array of lipids and membrane components. Analysis is typically accomplished by one of many histological methods, such as Hematoxylin and Eosin (H&E) stain, immunohistochemistry (IHC) and in situ hybridization (ISH). However, a limitation of most techniques is a requirement for prior knowledge of the targets of interest. Mass spectrometry (MS) coupled assays are useful for their inherent speed and accuracy. Hyphenated MS techniques, such as MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization-Time of Flight) have been developed for rapid analysis of complex biological samples. MALDI-TOF MS lends itself to tissue slices because it does not require pure samples and can offer de novo discovery of sample components. Here we show the coupling of this technique with histological staining for the investigation of lipids and their localization within mouse kidney tissue slices. This method is shown to be extensible through the incorporation of LIFT (MS/MS) wherein a specific peak of known molecular weight is exposed to a high energy laser which causes reliable and reproducible fragmentation based on bond energies within the molecule. As such, aspects of the target molecule from a class (eg phospholipids) down to side chains can be identified allowing the fullscale investigation of major tissue components. In a proof of concept study, pure standards of the major phospholipids phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) were subjected to LIFT, to confirm structures. Subsequently, MALDI-IMS applied to tissue slices reveals abundant peaks in the range of predicted phospholipids. These results will be analyzed to confirm these tissue phospholipids. MALDI-TOF MS coupled with LIFT presents a novel way of looking at tissue without prior knowledge of its constituents as it allows for analysis in the absence of traditional reagents such as antibodies or nucleic acid probes.