Fig 2: Arg2 TSB delivery decreases pro-inflammatory cytokines secretion in an LPS in vivo modelWT C57Bl/6J mice were given i.p. injection of Arg2 vs NC TSB for 24 h followed by LPS at 5 mg/kg (or PBS control) for 8 h. (A) Arg2 mRNA expression in PECs (left) and spleen (right). Data are presented as mean ± SEM and were compared by one-way ANOVA where mice injected with NC TSB and LPS were set at 1. (B) Arg2 and ß-actin in spleen. Blot representative of seven mice from each TSB injection (three of which were then injected with PBS and four with LPS for 8 h). (C) Pro-inflammatory cytokines levels in serum in the LPS model of acute inflammation. Data are presented as mean ± SEM and were compared by one-way ANOVA (Sidak’s multiple comparison test, *p < 0.05).

Fig 3: Arg2 TSB modulates surface markers and metabolic parameters in primary BMDM(A–D) Surface markers expression. Pro-inflammatory markers CD80 (A) and CD86 (B) and anti-inflammatory markers CD71 (C) and CD206 (D) surface levels in Arg2 vs NC TSB transfected BMDM (in presence and absence of LPS) were compared by flow cytometry (n = 3, in single). Histograms depict the results obtained in one representative experiment. The bar graphs represent the means of mean fluorescence intensity ± SEM of positive cells in three biological replicates. Data were compared by one-way ANOVA (Sidak’s multiple comparisons test, *p = 0.05, **p < 0.01, ****p < 0.0001). (E) Representative Seahorse metabolic flux trace of BMDM transfected with Arg2 vs NC TSB in absence (left) or presence (right) of LPS (10 ng/mL) (n = 5, 4–7 technical replicates). (F) Quantitative oxidative parameters changes in BMDM transfected with Arg2 vs NC TSB in absence or presence of LPS (n = 5, 4–7 technical replicates). Data were compared by two-way ANOVA (Tukey’s multiple comparisons test, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Fig 4: Arg2 TSB effectively blocks miR-155-mediated repression of Arg2Data are presented as mean ± SEM and were compared by two-way ANOVA (using multiple comparisons test, *p = 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (A) Visual map of the in silico predictions of miR-155 binding to its MRE within Arg2 3' UTR. (B) Arg2 3' UTR luciferase activity reported as percent change in relative light units (RLU) in RAW 264.7 cells. Site-directed mutagenesis was applied to pmir_Arg2_wt vector to disrupt the miR-155 MRE. RAW 264.7 cells were co-transfected with wt (first to fourth bars) or miR_155 mutant (fifth to eighth bars) plasmid and TSB and miR mimic (100 ng plasmid, 100 nM TSB, 25 nM mimic, n = 3, in triplicate). Samples co-transfected with WT plasmid, non-targeting control (NC) TSB and NC miR mimic were used as reference and set at 100%. For both WT and miR_155 mutant plasmids, only the comparison between NC and Arg2 TSB (co-transfected with NC or miR-155 mimics) are highlighted in the graph. (C) MiR-155 expression upon stimulation with LPS (100 ng/mL, 24 h) and TSB transfection in primary BMDM (n = 4, in triplicate). LPS-stimulated samples transfected with NC TSB are set as 1. (D) Arg2 TSB effect on endogenous levels of Arg2 mRNA in primary BMDM (n = 4, in triplicate). Unstimulated samples transfected with NC TSB are set as 1. (E) Arg2 TSB effect on endogenous levels of Arg2 protein in primary BMDM (n = 3, in single). LPS-stimulated samples transfected with NC TSB are set as 1. (F) Arginase activity assay in primary BMDM upon transfection of Arg2 TSB (n = 3, in triplicate). Urea was measured as a byproduct of arginase activity and fold arginase activity was measured by setting LPS-stimulated samples transfected with NC TSB as 1.