Fig 2: Model: CsA attenuates IAV-induced macrophages polarization and inflammatory responses by targeting CypA. CsA targets CypA to regulate IAV-triggered macrophages M1/M2 polarization by inhibiting STAT1 phosphorylation and promoting STAT6 phosphorylation, thereby suppressing inflammatory cytokines production and tissue injury.

Fig 3: IL-4 and IL-21 activated JAK/STAT pathways and stimulated downstream gene expressions in Ramos B cells. Ramos and Jurkat were treated with γc cytokines for 15 mins and harvested for western blot analyses. (A) IL-21 and IL-4 significantly induced STAT3 and STAT6 phosphorylation respectively. No STAT5 phosphorylation was observed in Ramos cells after any cytokine treatments. (B) IL-2, IL-9 and IL-21 treatments triggered STAT3 and STAT5 phosphorylation in Jurkat cells. Ramos cells were starved in serum-free RPMI1640 medium for 24 hours and treated with either IL-4 or IL-21 for 6 and 24 hours. (C) IL-4 significantly reduced CASP7 and induced CD23 expressions dose dependently at 6 and 24 hours respectively. (D) IL-21 significantly induced PRDM1 and IRF4 expressions dose dependently at 24 hours. *p < 0.05, **p < 0.01, ***p < 0.001 as compared to vehicle controls art respective time points by one-way ANOVA, N = 3.

Fig 4: Peimine relieves IL-4-induced M2 polarization via regulation of the STAT6 transcription factor, P38 MAPK, and Akt(A) The expression of p-STAT6, STAT6, p-p38 MAPK, p38 MAPK, p-Akt (s473), Akt in MH-S cells was evaluated by western blot. Left panel: Representative western blot results. Right panel: Bar graphs showing the mean data in each group. Data were expressed as means ± SEM (n=3). **P<0.01 vs. the normal group; #P<0.05 and ##P<0.01 vs. the model group. (B) The expression of p-STAT6 in MH-S cells was evaluated by immunofluorescence staining. Left panel: Immunofluorescence staining results (400×). Right panel: Bar graphs showing the mean data in each group. Data were expressed as means ± SEM (n=9). **P<0.01 vs. the normal group; ##P<0.01 vs. the model group.

Fig 5: TRPC1-/- mice show no M1 phenotype development even upon LPS treatment(A) Ca2+ imaging performed on M1 cells from control (WT) and TRPC1-/- mice. Representative trace showing Thapsigargin (Tg)-evoked [Ca2+]i changes in naive and M1 cells pretreated with and without LPS (100 ng/mL) treatment for 6 h. Data shown are representative of three independent experiments with 110–140 cells each.(B) Bar diagram showing quantification of the fluorescence ratio (340/380) from an average of ~100 cells are shown underneath the traces. Bar graphs depict average ± SEM (Student's t test). Data shown are representative of three independent experiments with similar results.(C and D) Application of 2 µM Thapsigargin (Tg) in bath solution induced inward currents at -80 mV in control and LPS (100 ng/mL)-treated M1 cells. Respective IV curves of currents and quantitation (8–10 recordings) of current intensity at -80 mV are shown in (C). Bar graphs depict average ± SEM (Student's t test). Data shown are representative of three independent experiments with similar results.(E) Ca2+ imaging performed in M2 cells from control (WT) and TRPC1-/- mice. Representative trace showing Thapsigargin (Tg)-evoked [Ca2+]i changes in naive and M2 treated cells upon pretreatment with and without LPS (100 ng/mL) for 6 h. Data shown are representative of three independent experiments with similar results.(F) Bar diagram showing quantification of the fluorescence ratio (340/380) from an average of 90–120 cells are shown underneath the traces. Bar graphs depict average ±SEM, *p = 0.05 (Student's t test). Data shown are representative of three independent experiments with similar results.(G and H) Application of 2 µM Thapsigargin (Tg) in bath solution induced inward currents at -80 mV in control and LPS (100 ng/mL)-treated M2 cells. Respective IV curves of currents and quantitation (5–8 recordings) of current intensity at -80 mV are shown in (G). Bar graphs depict average ± SEM, *p = 0.05 (Student's t test). Data shown are representative of three independent experiments with similar results.(I) Heatmap showing Log2 Fold changes (N = 3) in selected M1-inflammatory and M2-anti-inflammatory genes in bone marrow-derived macrophages from WT mice treated with IFN? at 20 ng/mL over medium alone (NS), and corresponding cells from TRPC1-/- mice cultured in presence of IFN? are shown in columns 1 and 2, respectively.(J) A 2D scatter plot showing comparison as in (I). The x axis shows Log2 fold change values of IFN?-treated WT macrophages over unstimulated samples. The y axis shows Log2 fold change values of IFN?-treated TRPC1-/- over unstimulated TRPC1-/- samples. The color of dots represents statistical significance in differential gene expression. Genes with an adjusted p value < 0.05 were considered as differentially expressed. Red dots denote differentially expressed genes on both comparisons, whereas green dots represent genes that are significant in IFN? versus NS. Genes differentially expressed in TRPC1-/- (NS versus treated) are shown as purple dots and those non-significant in both conditions are represented by blue dots.(K) Box plots showing Log2 fold change of the expression of pro- and anti-inflammatory genes in IFN? treated over unstimulated WT and TRPC1-/- macrophages are shown in boxes 1 and 2, respectively from three independent experiments.(L) Flow cytometry was performed to measure the difference between wild type and TRPC1-/- of classic internal M1 markers (iNOS+) and M2 markers (Arg1+) and surface M1 (CD80+) and M2 (CD206+) markers. Data shown are representative of three independent experiments with similar results.(M) Macrophage (M0, M1, and M2) cells from TRPC1-/-mice cultured in 6-well plates (2 × 106 cells/well) and treated with or without LPS (100 ng/mL) for 6 h. The concentration of pro-inflammatory (IL-6 and IFN?) cytokines released in the media was performed by ELISA. Data shown are representative of three independent experiments with similar results. Bar graphs depict average ± SD for relative values, NS, non-significant (Student's t test).(N) Immunoblotting analysis showing the level of expression of wild-type mice versus TRPC1-/- M0, M1, and M2 BMDM polarization pathway genes pNF-?B, NF-?B, pJNK, JNK, pSTAT6, STAT6, pSTAT1, and STAT1 and ß-actin. Data shown are representative of three independent experiments with similar results.