MSACL 2018 US Abstract

Topic: Tissue Imaging & Analysis

Assessing the Effect of Immunization on the Global Lipidome of Mouse Spleen Using MALDI FTICR IMS

Marissa Jones (Presenter)
Vanderbilt University

Bio: Marissa received her B.S. in Chemistry from Southwest Baptist University (SBU) in 2016. During her undergraduate studies, she completed two summer research experiences. The first was a Summer Undergraduate Research Fellowship (SURF) internship at SBU in physics, which focused on instrumentation. The second was a Research Experience for Undergraduates (REU) at the University of Missouri. It focused on inorganic and organic chemistry and the application of polyhedral boranes to cancer therapeutics. Marissa’s current graduate work focuses on the application of Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI IMS) to biological questions. Currently, she is using MALDI IMS to probe immunological responses within spleen as well as studying the effect of sample preparation on the global lipidome.

Authorship: Marissa Jones (1,2), Nathan Heath Patterson (1,3), William Perry (1,2), Sung Hoon Cho (4), Mark R. Boothby (4,5,6,7,8), Jeffrey Spraggins (1,2,3), Richard M. Caprioli (1,2,3,5,8)
(1) Mass Spectrometry Research Center, (2) Department of Chemistry, Vanderbilt University, Nashville, TN, (3) Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, USA, (4) Department of Pathology, Microbiology and Immunology, School of Medicine, Vanderbilt University, and Vanderbilt University Medical Center, Nashville, Tennessee, USA, (5) Department of Medicine, Vanderbilt University, Nashville, Tennessee, USA, (6) Department of Cancer Biology, Vanderbilt University, Nashville, Tennessee, USA, (7) Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee, USA, (8) Department of Pharmacology, Vanderbilt University, Nashville, Tennessee, USA

Short Abstract

Hypoxia plays a key role in immune response and can have mechanistic downstream effects on B cell survival, proliferation, and differentiation. Germinal Center (GC), where B cell undergoes proliferation, selection and differentiation, consists of light and dark zones in which the native oxygen levels vary. Immunization increases the size and number of GCs. While the connection between hypoxia and inflammation is widely understood, the effect of these hypoxic regions on biomolecular modification and distribution within activated lymphoid tissue structures are largely uncharacterized. Herein we propose using high mass and spatial resolution MALDI FTICR IMS to examine the effect of immunization on the differential expression of biomolecules within spleen.

Long Abstract

Introduction

Hypoxia, the lack of oxygen, is often a key aspect in a variety of diseases and immune responses.[1] For example, hypoxia is relevant to a variety of cellular mechanisms such as angiogenesis, inflammation, apoptosis, cartilage degradation, energy metabolism and oxidative damage.[2] Mechanistically, hypoxia can produce a variety of downstream effects. Restriction of oxygen tension in Germinal Centers (GCs) has led to a decrease in B cell proliferation that, in turn, causes stricter survival signal thresholds.

Although the link between hypoxia and a variety of cellular mechanisms is well understood, the microenvironments of the oxygen landscape within lymphoid tissue structures, specifically GCs are largely uncharacterized.[3,4] GCs are sites of antibody class-switch recombination and affinity maturation that are activated during an immune response.[5] GCs consist of two main zones, a dark zone in which B cell proliferation takes place, and a light zone in which B cell diversification through antibody class switching and affinity maturation occurs.[4,5] In mice, GC light zones are hypoxic, reducing B cell proliferation and impairing antibody class switching.[3] It is hypothesized that these light and dark zones within germinal centers have unique biomolecular constituents and distributions driving changes in GC function.

Herein, we propose to utilize MALDI IMS to investigate the effect of immunization on GC biomolecules. MALDI IMS is specifically suited for this study for two main reasons: (1) the ability to directly compare IMS data to histological data, and (2) the application to untargeted molecular studies. GCs are approximately 200-400 micrometers in diameter, making them targetable with a wide variety of instrumentation.[6] With these abilities in mind, we will examine the effect of immunization on the differential expression of biomolecules within spleen.

Methods

C57/BL6 mice were euthanized at 7 days post immunization by extended exposure to isoflurane in accordance with the Vanderbilt Institutional Animal Care and Use Committee. Mouse spleen was embedded in 2.6% carboxymethyl cellulose sodium salt, frozen on dry ice, and sectioned at 7 µm. Samples were sublimated with 1,5-daiminonapthalene and lipidomic data was acquired with a 9.4T Bruker SolariX FTICR mass spectrometer (Bruker Daltonics, Billerica, MA, USA). Preliminary data was collected in negative ion mode from m/z 250-2000 with a raster step of 30 µm on one half of a spleen and in positive mode on the other half from m/z 500-2000. Imaging data were collected using 500 laser shots (Laser repetition rate: 2 kHz) per pixel with a laser beam focused to ~20 µm. All data was acquired with a mass resolving power of ~82,000 at m/z 400. Internal Lock mass calibration was performed during imaging experiments using known lipid species: Phosphatidylinositol (38:4) at m/z 885.5490 in negative mode and Phosphatidylcholine (32:0) at m/z 734.5694 for positive mode analysis.

A novel data analysis pipeline was developed to identify lipids based on accurate mass and to visualize global changes in intensity. A custom augmented version of lipid maps was imported into SCiLS as a FlexImaging result list and Receiver Operating Characteristic (ROC) discriminate peaks were found for immunized spleens versus control spleen. ROC values, as well as corresponding intensities were exported for further processing and visualization using R to visualize global trends. The ROC values provided a quantitative measure for the level of discrimination of each peak. These differentially expressed ions were then further investigated for ion localization in both SCiLS and FlexImaging 5.0.

Results

A variety of lipids are shown to have differential expression and localization within immunized and non-immunized control spleen samples, and have been punitively identified based on accurate mass measurements. Ions at m/z 691.48 (negative ion mode) and 837.63 (positive ion mode) were tentatively identified to be phosphatidylinositol (PI(36:3)) and phosphatidic acid (PA(44:1)), respectively, and both were found to be localized to substructures within white pulp. Their size and distribution are consistent with the size and distribution of GCs and possibly their substructures. Upon comparison of non-immunized control and immunized spleen, the presence of these small tissue sub-regions are greater in the immunized samples, suggesting localization to germinal centers and their substructures. Ions at m/z 746.57 and 835.62 have been tentatively identified as phosphatidylethanolamine (PE(36:1)) and PA(44:2), respectively. Both of the lipids show distributions to all surrounding sub-regions of tissues, except for the GC-like substructures within white pulp. These identifications were made in both positive and negative mode, confirming the viability of FTICR Imaging Mass Spectrometry to investigate the GCs and possibly their substructures within immunized and non-immunized control spleen samples.

Through the application of a novel data analysis pipeline, ROC heat maps were created to better visualize global trends within the data. In general, shorter chain length phosphatidylethanolamines (PEs) and glycerophospholipids were found to have widely varying ROC values than their longer chain counter parts in negative ion mode. We have found that the differentiation in PEs is highlighted by PE(O-34:2) and LysoPE(16:0), which suggest molecular effects that characterize immunized tissues when compared to non-immunized control tissues due to their ROC values. PE(O-34:2) has an ROC value of 0.261 indicating its ability to characterize immunized tissue and LysoPE(16:0) has an ROC value 0.734 indicating its ability to characterize non-immunized control tissue.

Also, for glycerophospholipids, PA(20:1) was found to be a better indicator for non-immunized control tissue and PA(32:1) for immunized tissue with ROC values of 0.858 and 0.307 respectively.

Conclusions & Discussion

In conclusion, through the use of a novel pipeline and high resolution MALDI FTICR IMS, biomolecular changes within GCs can be explored. Immunized mice show a distinct difference in both localization and abundance of many lipids possibly corresponding to the GCs and their light and dark zones within spleen tissue. Immunized mice also show a global increase in longer chain phosphatidylethanolamines and glycerophospholipids relative to their non-immunized counter parts.


References & Acknowledgements:

References:

1. Eltzschig, H. K.; Carmeliet, P., Hypoxia and Inflammation. N Engl J Med 2011, 364 (7), 656-65.

2. Quiñonez-Flores, C. M.; González-Chávez, S. A.; Pacheco-Tena, C., Hypoxia and its implications in rheumatoid arthritis. In J Biomed Sci, 2016; Vol. 23.

3. Cho, S. H.; Raybuck, A. L.; Stengel, K.; Wei, M.; Beck, T. C.; Volanakis, E.; Thomas, J. W.; Hiebert, S.; Haase, V. H.; Boothby, M. R., Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response system. Nature 2016, 537, 234-238.

4. Konisti, S.; Kiriakidis, S.; Paleolog, E. M., Hypoxia--a key regulator of angiogenesis and inflammation in rheumatoid arthritis. Nat Rev Rheumatol 2012, 8 (3), 153-62.

5. Allen, C. D.; Okada, T.; Cyster, J. G., Germinal Center Organization and Cellular Dynamics. Immunity 2007, 27 (2), 190-202.

6. Victora, G. D.; Schwickert, T. A.; Fooksman, D. R.; Kamphorst, A. O.; Meyer-Hermann, M.; Dustin, M. L.; Nussenzweig, M. C., Germinal center dynamics revealed by multiphoton microscopy with a photoactivatable fluorescent reporter. Cell 2010, 143 (4), 592-605.

Acknowledgments:

This work was supported by NIH 5P41GM10339107 Grant and NIGMS 1S100D012359 Shared Instrumentation Grant.


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