Fig 2: The influence of S100A9 in LoVo-induced MDSCs vitality, chemotaxis and activation in vitro. (A) The CD33+ cells were separated from PBMCs with CD33 positive magnetic beads, and co-cultured with LoVo cells to induce into CRC-associated MDSCs in vitro. (B) Identification of LoVo induced-MDSCs characterized by the myeloid marker with HLA-DR-CD33+CD11b+ by FCM. CD33+ cells cultured in medium alone were included as a control. (C) CCK8 assay for cell viability of LoVo-induced MDSCs treated with different concentrations of GST-S100A9 (5, 10, and 20 µg/ml) and GST proteins for 12, 24, and 48 h. (D) Chemotaxis assay for LoVo-induced MDSCs treated with different concentrations of GST-S100A9 (5, 10, and 20 µg/ml) and GST proteins for 24 h. (E–G) Real-time PCR analysis for mRNA expression of immunosuppressive molecules, including Arg-1 (E), iNOS (F), and IL-10 (G) in LoVo-induced MDSCs after GST-S100A9 (20 µg/ml) and GST (20 µg/ml) protein treatment for 24 h. (H) Fluorescence intensity analysis for ROS production in LoVo-induced MDSCs treated with GST-S100A9 (20 µg/ml) and GST (20 µg/ml) proteins. (I) Suppression of LoVo-induced MDSCs treated with GST-S100A9 (20 µg/ml) and GST (20 µg/ml) proteins on T cells in vitro. (J) A statistical graph of the suppressive effect of LoVo-induced MDSCs treated with GST-S100A9 (20 µg/ml) and GST (20 µg/ml) proteins on CD8+ T cells. Data represent three independent experiments and are represented as Mean±SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Imipramine downregulates an M2-like program in TAMs(A) Western blot analysis of ARG1 and IL-10 in single tumors treated or not with IM or anti-VEGF.(B) Flow cytometry analysis of ARG1 and IL-10 expression in GBM tumors treated for 1 week. Ctrl (n = 9 tumors), IM (n = 6), anti-VEGF (n = 6), anti-VEGFR2 (n = 6), Axitinib (n = 6). Macrophages were gated as CD45+CD11b+Ly6C-Ly6G-.(C) Expression of ARG1 and IL-10 in microglia (CD49d-) and MDMs (CD49d+) assessed by FACS in untreated tumors (n = 5).(D) Expression of MHC-II within microglia as assessed by flow cytometry. Ctrl (n = 4 tumors) and IM + anti-VEGF (n = 4).(E) Ex vivo co-cultures of tumoral CD11b cells and activated splenic CFSE-labeled CD8 or CD4 T cells. Each dot represents the average of two or three technical replicates. T cells alone (n = 4), Ctrl co-culture (n = 5), anti-VEGF (n = 3), IM (n = 4), IM + anti-VEGF (n = 4).(F) Analysis of the M2-like program in cytokine-polarized macrophages as assessed by qRT-PCR analysis of Ctrl and IM-treated M2-like BMDMs. Expression is normalized to 18S statistics by Welch’s t test. Each dot represents an individual sample. Data are representative of three independent experiments.(G) Analysis of the M1-like program in BMDMs assessed by FACS. Each dot represents an individual replicate.(H) Expression of Hrh1 mRNA normalized to 18S in ex vivo M1-and M2-polarized BMDMs, either untreated or IM treated for 24 h. Each dot represents an individual sample. Data are representative of three independent experiments.(I) mRNA expression of Hrh1 in FACS-sorted microglia or MDMs from Ctrl and IM-treated tumors.(J) mRNA expression of Arg1, Chil3, and Il10 in M2-polarized macrophages that were transfected with siCtrl or two different siHrh1 constructs. Cells were treated with 40 µm IM for 24 h. Data are representative of two independent experiments.(K) Western blot analysis of MRC1 and ARG1 expression of siRNA-transfected M2 BMDMs. Data are representative of two independent experiments.(L) CD8 and CD4 T cell proliferation during co-culture with tumoral CD11b cells isolated from untreated (n = 2) or TFP-treated tumors (n = 4).(M) mRNA expression of Hrh1, Arg1, and MMP2 in CD11b cells isolated from tumors treated with IM (n = 4), TFP (n = 4), or untreated Ctrl (n = 5).(N) Phagocytosis assay involving sorted microglia and MDMs from untreated or IM-treated tumors assayed with green pHrodo S. aureus bioparticles. Data presented as mean fluorescence intensity (MFI) of pHrodo/live cells. (Para break) Data in all quantitative panels are presented as mean ± SEM *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, no statistical significance. Statistical analysis by Mann-Whitney test or one-way ANOVA, unless otherwise stated.

Fig 4: ST258 Klebsiella pneumoniae strains induced M(Kp) polarisation AImmunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected with different K. pneumoniae strains, Kp52145, NJST258-1, NJST258-2 or SHG10, for 60 min.BImmunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected with Kp52145 or KP35 for 60 or 120 min.C, DAnalysis by flow cytometry of the levels of M(Kp) markers expressed by IMs and AMs from PBS-mock-infected mice (black dots), and by IMs and AMs from infected mice (purple dots) with and without associated KP35 24 h post infection. (C) Percentage of positive cells for Arg1. (D) Percentage of positive cells for CD206.EImmunoblot analysis of Arg1 and tubulin levels in lysates from wild-type and stat6 -/- iBMDMs non-infected (ni) and infected with KP35 for 60 min or 120 min.FPercentage of wild-type (WT) and stat6 -/- iBMDMs with and without associated KP35 positive for CD206 5 h post infection. KP35 was tagged with mCherry.GPercentage of wild-type (WT) and stat6 -/- iBMDMs with and without associated KP35 positive for MCH-II 5 h post infection. KP35 was tagged with mCherry. Data information: For all infections, after 1 h contact, medium replaced with medium containing gentamycin (100 µg/ml) to kill extracellular bacteria. Images are representative of three independent experiments. Error bars are presented as the mean ± SEM of three independent experiments in duplicate. Statistical analysis were carried out using one-way ANOVA with Bonferroni contrast for multiple comparisons test. ****P = 0.0001; **P = 0.01; ns, P > 0.05 for the indicated comparisons.

Fig 5: Klebsiella pneumoniae induces M(Kp) polarisation in human macrophages arg1 mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. il10 mRNA levels were assessed by qPCR in M-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. chi3l mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h. ido mRNA levels were assessed by qPCR in hM-CSF-treated PBMCs from 6 donors non-infected (ni) or infected Kp52145 for 1, 3 or 5 h.Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 for 60 or 120 min. arg1 mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with the STAT6 inhibitor AS1517499 or DMSO vehicle control. il10 mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with the STAT6 inhibitor AS1517499 or DMSO vehicle control. nos2 mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with the STAT6 inhibitor AS1517499 or DMSO vehicle control. isg56 mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with the STAT6 inhibitor AS1517499 or DMSO vehicle control. ido mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with the STAT6 inhibitor AS1517499 or DMSO vehicle control. arg1 mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with IFNAR1 blocking antibody or isotype control. ido mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with IFNAR1 blocking antibody or isotype control. arg1 mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with IL10 blocking antibody or isotype control. ido mRNA levels were assessed by qPCR in PMA-treated THP-1 macrophages non-infected (ni) or infected Kp52145 for 1, 3 or 5 h and treated with IL10 blocking antibody or isotype control.Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from PMA-treated THP-1 macrophages non-infected (ni), or infected with Kp52145 or the CPS mutant, strain 52145-?wca K2 , for 60 or 120 min.Percentage of PMA-treated THP-1 macrophages with and without associated Kp52145 or 52145-?wca K2 positive for Arg1 5 h post infection. Bacteria were tagged with mCherry.Percentage of PMA-treated THP-1 macrophages with and without associated Kp52145 or 52145-?wca K2 positive for CD206 5 h post infection. Bacteria were tagged with mCherry. Data information: For all infections, after 1 h contact, medium replaced with medium containing gentamycin (100 µg/ml) to kill extracellular bacteria. Error bars are presented as the mean ± SEM of three independent experiments in duplicate. Images are representative of three independent experiments. Statistical analysis were carried out using one-way ANOVA with Bonferroni contrast for multiple comparisons test. ****P = 0.0001; ***P = 0.001; **P = 0.01; *P = 0.05; ns, P > 0.05 for the indicated comparisons.