Understanding the link between an immune response and the changing metabolic states of cells may provide opportunities to correct defects seen in many inflammatory diseases. Characterizing the oxygen tension among the different regions of the lymph node as they go through various levels of immune response may lead to a deeper understanding of their respective metabolic processes as oxygen consumption is directly related to the metabolic state of a cell. However, there are few simple methods for spatially resolved optical measurement of local oxygen tension in live tissue samples. Here, we took advantage of oxygen sensing boron nanoparticles (BNPs), previously described by the Fraser lab, to investigate oxygen tension in live murine lymph node tissue under various states of immune response. BNPs provide ratiometric quantification of local oxygen tension based on oxygen-independent fluorescence and oxygen-quenched phosphorescence, and can be imaged using a camera assembly attached to a microscope for high resolution detection. Two systems were tested for the sue of the BNPs. First, BNPs were loaded into the tissue by gentle centrifugation. Second, BNPs were held in close proximity to the tissue on a culture surface. The viability of the lymph node slices after exposure to BNPs in either format was confirmed by dual calcein/PI staining (live/dead assay). The BNPs were shown to be non-toxic to the tissue slices. Next, we tested whether the previously characterized oxygen sensitivity of the BNPs was replicated in tissue slices. Lymph node slices were exposed to various concentrations of oxygen, and ratiometric imaging was performed. BNPs embedded in the tissue or layered on top of the lymph node slice both exhibited lower levels of phosphorescence when exposed to increased oxygen concentrations. This resulted in higher observed fluorescence/phosphorescence ratios for those areas of higher oxygen tension. Optimization of the uniformity of the BNP in this assay is being conducted, to ensure even distribution across the tissue. In future work, the change in oxygen tension after stimulation with an immunogenic agent will be investigated. We anticipate that this easy-to- use assessment of oxygen tension may be used as one measure of metabolic state in the lymph node and other soft tissues.

Utilizing oxygen sensing boron nanoparticles to develop a novel immunometabolism assay

254th National Meeting (2017)

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