Fig. 5: Live neutrophil trapping disrupts anti-inflammatory signaling and influences the tissue milieu.

A Schematic of the experimental design was created using Biorender.com: Peritonitis was induced in WT mice with an i.p. injection of 5 mM sodium metaperiodate. 72 h after the onset of peritonitis, 10⁷ apoptotic or RgpB-treated murine bone marrow neutrophils (a-BMN and g-BMN, respectively) from CD45.1 donor mice were labeled with CellTrace Violet and injected into the inflamed peritoneal cavities of CD45.2 recipient mice. 4 h post-injection, peritoneal exudate macrophages (PEMs) were sorted into two populations of phagocytosing macrophages or ‘bystander’ macrophages based on Cell trace positivity using the gating strategy described in Fig. S5B. Sorted cells were analyzed by RNA-seq. B PCA plot depicting clustering of PEMs based on transcriptional responses of peritonitis mice that did not receive g-BMN or a-BMN injection (PEM), g-BMN or a-BMN ingesting macrophages, or ‘bystander’ macrophages from g-BMN or a-BMN injected mice (g-BMN ctrl and a-BMN ctrl, respectively). C Bar plot showing enriched catabolism & autophagy, inflammation, and metabolism pathways. Bars to the left depict upregulated pathways in g-BMN ingesting macrophages; bars to the right depict upregulated pathways in macrophages that ingested a-BMNs. Statistical significance can be found in. D, E Heatmaps showing significant differentially expressed inflammatory genes, lysosomal genes, and lipid catabolism genes in g-BMN vs a-BMN ingesting macrophages (D) or control non-effercytosing or bystander macrophages from same mice (E). Statistical significance amongst represented genes can be found in Supplementary Tables 7, 9. F Heatmap depicting significant differentially expressed inflammatory genes in a-BMN ctrl vs g-BMN ctrl macrophages. Pink indicates highly expressed genes. Statistical significance amongst represented genes can be found in Supplementary Table 8. Oral inflammation was induced in mice by placing ligatures on the second maxillary molars of wildtype mice as depicted in (F). On alternate days, mice received sterile saline (denoted as ‘Lig only’) or 10⁷ g-BMN via tail vein injection (i.v.). On day 7, mice were euthanized, and alveolar bone loss was assessed by μCT. G Representative μCT images showing alveolar bone recession surrounding ligature sites M2 and M2’ molars. H, I Quantification of M2 and M2’ bone loss is shown in mm. Averaged data from 7-8 mice is shown as mean ± SD, and statistical significance determined using an unpaired t-test; *p < 0.05, **p < 0.02.