N-glycan signature of neutrophil function in a Staphylococcus aureus skin infection model by mass spectrometry imaging

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Abstract Staphylococcus aureus (Sa) is a public health concern due to the rise of antibiotic-resistant strains. In humans, elevated risk is associated with Tumor Necrosis Factor (TNF) inhibition. Neutrophils expressing TNF receptors (TNFR1 and TNFR2) respond to Sa skin infection in a mouse model. TNFR2 activation induces PAD4 (peptidylarginine-deiminase-4) which leads to neutrophil extracellular traps (NET) synthesis, increasing host defenses. Alterations in PAD4 and N-glycans are related to increased skin infections and the spatial glycosylation signature during the skin response to Sa is unknown. We compared N-glycan signatures in neutrophil region between wildtype (WT), TNFR1 KO, TNFR2 KO and PAD4-/- groups. Mice were intradermally injected with Sa and skin samples collected for analysis by mass spectrometry imaging. We sprayed PNGaseF on prepared FFPE sections to cleave the N-glycans, sprayed with CHCA matrix, and evaluated in positive ion mode at 50mm spatial resolution on a timsTOF Flex (Bruker Daltonics). Differential N-glycan signatures were identified using a histology-guided approach limiting the comparisons to the PMN region. PAD4-/- showed higher intensity of N-glycans than all groups. However, specific N-glycans were higher in TNFR1 KO than PAD4-/- and TNFR2. A lower profile was found in TNFR2 KO vs. WT. We concluded that each group has a specific N-glycan signature which can be attributed to unique immune responses to Sa infection and NET formation.