Exploring the host-pathogen response within the TB granuloma microenvironment using mass spectrometry imaging
Claire L. Carter
Center for Discovery and Innovation, Hackensack Meridian Health, Nutley, New Jersey, 07110, United States
It is known that macrophage plasticity and phenotype within the granuloma environment are critical to either tuberculosis control or progression. How these dichotomous pathways evolve during the course of infection remain to be elucidated. The aim of this study is to define the biochemical and metabolic status of the host cell subsets at the spatial level within the developing granuloma. Mass spectrometry imaging is a label-free and untargeted technique, providing the opportunity to simultaneously identify and spatially resolve energy pathway metabolites and signaling lipids within the histologically well characterized granulomas.
Rabbits were infected with Mtb strain HN878 for 16-20 weeks, at which point the lungs have developed the spectrum of granulomas observed in humans. Upon euthanasia, lungs were resected and cellular, early-necrotizing, necrotic and cavity containing lesions were dissected and snap frozen. For sterilization, samples were gamma irradiated whilst frozen and then transferred to a -80C freezer until use. Lesions were serially sectioned at 10 µm and transferred to positively charged glass slides for histology and MALDI-MSI. For lipid and metabolite analysis 9-AA was deposited over the slides using the HTX sprayer. MSI analysis was carried out on a Bruker Solarix XR 7T mass spectrometer and imaging data were processed using FlexImaging and the SCiLs lab software (Bruker Daltonics, Massachusetts, USA). Following MSI the samples were washed and stained with H&E. The MSI data was then directly overlaid with its corresponding stained section for data analysis and co-registration of ion distribution with pathology.
Our preliminary data detected an accumulation of several pleiotropic sphingolipids, including ceramide-1-phosphates (C1P), within the macrophage and necrotic regions of each lesion. These preliminary results demonstrated phenotypical differences in macrophages based on the fatty acid chain length and number of double bonds in the C1P structure. We simultaneously detected metabolites involved in glycolysis and the pentose phosphate pathway, and a number of mitochondrial specific lipids that also displayed a differential distribution within histologically similar host cells. This was evident in the imaging data as neighboring macrophages displayed differences in the intensity and detection of distinct lipids and metabolites. These differences also correlated to the stage of infection or lesion formation.
Ceramide-1-phosphate is a pleotropic sphingolipid that regulates macrophage function in a manner that mirrors those observed during Mtb infection. These include the inhibition of apoptosis, increased cell cycle and proliferation, macrophage recruitment/migration and immune cell signaling. Additionally, alveolar and interstitial macrophages are known to be restrictive or permissive to bacterial growth and this has been linked to their metabolic status. Our preliminary results spatially resolved lipids and metabolites within different TB lesions and determined differences related to macrophage phenotype, metabolic status and related signaling lipids. These data provided spatially resolved biochemical alterations during lesion development and progression, identifying targets for future mechanistic studies and new therapeutic strategies related to lipid signaling and macrophage phenotype.