Animal study overview, clinical and immunological results. A: Study overview, preterm pigs were nourished with parenteral nutrition (PN) with either high (21%, 30 g/kg/d) or low (5%, 7.2 g/kg/d) glucose supply and infused with either Staphylococcus epidermidis or control saline. Animals were followed for 22 hours and blood samples collected for further analysis. Figure created with BioRender.com. B: Survival of animals during experiment, presented as time to euthanasia according to predefined humane endpoints with corresponding log-rank test comparing SE-High and SE-Low C: Blood glucose, measured by glucose meter 3, 6 and 12 hours after SE inoculation as well as at euthanasia, presented as 95% box plots. D,E: Blood gas parameters collected before inoculation with SE (0 hours) and 3, 6 and 12 hours after as well as at euthanasia, presented as 95% box plots. F: Blood bacterial density in infected groups at 3, 6, 12 and 22 hours after bacterial inoculation, presented as 95% box plots on a logarithmic scale. G-K: Blood hematology and plasma cytokines at 3, 6, 12 and 22 hours after bacterial inoculation, presented as 95% box plots. C-K: Data at each time point analyzed separately, bars labeled with different letters are significantly different from each other (P < 0.05), n = 8-9 for control animals, and 10-16 for infected groups.

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Impacts of glucose supply on liver transcriptomics at euthanasia. A: principal component analysis plot of transcriptomic profiles among the four groups. B: Heatmap of differentially expressed genes (DEGs) related to glycolysis/gluconeogenesis, in the enriched pathways between the two infected groups. Differences shown as Z-scores, where red color indicates a higher expression and blue a lower. C: Gene set enrichment analysis (GSEA) with gene ontology database showing the top pathways activated and suppressed by reduced glucose supply in infected animals. Size of dots indicates number of DEGs while red color indicates lower adjusted P value. D-G: Expression of genes exclusively related to glycolysis or gluconeogenesis, shown as relative expressions using 95% box plots. *: P < 0.05, ***: P < 0.001, n = 7-8 for each control group and 15-16 for infected groups.

Impacts of glucose supply on plasma metabolism response revealed by proton NMR-based metabolomics at euthanasia, either at humane endpoint or 22 hours after bacterial inoculation. A: Score plot of principal component analysis performed on metabolomics data among the four groups. B: Volcano plot showing the differential expressed metabolites (DEMs) in SE-Low vs SE-High. Yellow color indicates higher plasma levels, red lower. C: Heatmaps of identified DEMs between the two infected groups. Differences shown as Z-scores, where red color indicates a higher expression and blue a lower. D: Schematic representation of gluconeogenesis and TCA cycle metabolites altered between the two infected groups. Red represents a metabolite upregulated in SE-Low vs SE-High whereas dark blue indicates a metabolite downregulated; light blue indicates a metabolite not detected by 1H NMR. E-G: Plasma alanine transaminase (ALT), aspartate transaminase (AST), and BUN levels, shown as 95% box plots. H-L: DEMs involved in gluconeogenesis and TCA cycle, shown as 95% box plots. *: P < 0.05, **: P < 0.01, ***: P < 0.001, n = 7-8 for each control group and 15-16 for infected groups.

Impacts of glucose supply on plasma proteome at different time points post-bacterial infection. A-D: Score plot of principal component analysis of proteome profiling changes from 3 hours to euthanasia among the four groups. E-F: Top pathways differing between SE-Low and SE-High at 12 hours post bacterial inoculation or euthanasia, were enriched using DAVID. Involved differential expressed proteins (DEPs) counts displayed on X axis and size of dots indicates number of DEPs, while red color indicates lower P value. G-I: DEPs involved in glycolysis or gluconeogenesis, including Lactate Dehydrogenase B (LDHB), Aldolase, Fructose-Bisphosphate A (ALDOA), Dihydrolipoamide dehydrogenase (DLD. J-L: DEPs of porcine IAIP related proteins, heavy chains 1 and 2 (ITIH1, ITIH2) and Alpha-1-Microglobulin bikunin precursor (AMBP). G-L: Shown as relative abundances with 95% box plots, data analyzed separately for each timepoint, bars labeled with different letters are significantly different from each other (P < 0.05), n = 6 in each group.

Impacts of glucose supply on whole blood transcriptomics at 12 hours after bacterial inoculation. A: Score plot of principal component analysis of transcriptomic profiles performed among the four groups. B, C: Gene set enrichment analysis (GSEA) of the immune response signaling (GO:0002764) with negative enrichment score and glycerolipid metabolic process (GO:0046486) with positive enrichment score in the SE-Low, relative to SE-High animals. D: Enrichment analyses using GSEA with gene ontology database showing the top pathways activated and suppressed by reduced glucose supply in infected animals. Size of dots indicates number of DEGs while red color indicates lower adjusted P value. E, F: Heatmaps of differentially expressed genes (DEGs) involved in the enriched immune response and glycerolipid metabolism pathways between the two infected groups. Differences shown as Z-scores, where red color indicates a higher expression and blue a lower. n= 6 in each control group and 13-14 for infected groups.

Follow-up experiment investigating effects of changing glucose regimen during infection. A: Study overview, preterm pigs were all started on high glucose regimen and inoculated with S. epidermidis. After 3 hours one group was shifted to low glucose PN (SE-3h), while after 6 hours another group was similarly shifted (SE-6h) and the remaining pigs continued on high glucose PN for the rest of the experiment (SE-12h). Figure created with BioRender.com. B: Survival during experiment, presented as Kaplan-Meier curves. C, D: Blood gas data collected at baseline and 3-12 hours after inoculation. E: Blood bacterial density 3-12 hours after inoculation, shown on a logarithmic scale. F: Blood glucose levels 3-12 hours after inoculation. G-I: Plasma cytokine levels 3-12 hours after inoculation. C-I: Presented as 95% box plots, data at each time point analyzed separately, bars labeled with different letters are significantly different from each other (P < 0.05), n = 11-14 for SE-12h, n = 12-15 for SE-3h and n = 10-12 for SE-6h.

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Human IAIP intervention, clinical and immunological results. A: Study overview, preterm pigs were nourished low (5%, 7.2 g/kg/d) glucose parenteral nutrition and infused with either Staphylococcus epidermidis or control saline. At one and 12 hours post inoculation, infected animals were treated with either human IAIP (50mg/kg) or saline and followed for 22 hours while blood samples collected for further analysis. Figure created with BioRender.com. B: Survival of animals during experiment, presented as time to euthanasia according to predefined humane endpoints with corresponding log-rank test comparing SE-Low and SE-IAIP C: Blood glucose, measured by glucose meter 3, 6 and 12 hours after SE inoculation as well as at euthanasia, presented as 95% box plots. D,E: Blood gas parameters collected before inoculation with SE (0 hours) and 3, 6 and 12 hours after as well as at euthanasia, presented as 95% box plots. F: Blood bacterial density in infected groups at 3, 6, 12 and 22 hours after bacterial inoculation, presented as 95% box plots on a logarithmic scale. G: Plasma IL-10 and blood neutrophil fraction at 3, 6, 12 and 22 hours after bacterial inoculation, presented as 95% box plots. H: I: Cord blood neutrophil phagocytic rate and capacity following in vitro challenge with fluorescently labeled E. coli and treatment with increasing doses of IAIP, n = 7. C-H: Data at each time point analyzed separately, bars labeled with different letters are significantly different from each other (P < 0.05), n = 8 for control animals, and 10-18 for infected groups.

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