Fig 2: 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.

Fig 3: Klebsiella pneumoniae exploits type I IFN signalling to induce M(Kp) polarisation Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1 -/- for 60 or 120 min.Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or ifnar1 -/- for 60 or 120 min. arg1 mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1 -/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h. nos2 mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1 -/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h. pparg mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1 -/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h. fizz1 mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1 -/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h. il10 mRNA levels were assessed by qPCR in wild-type (WT), irf3 -/-, ifnar1 -/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.Percentage of wild-type (WT) and ifnar1 -/- iBMDMs with and without associated Kp52145 positive for Arg1 1, 3 or 5 h post infection. Kp52145 was tagged with mCherry.Percentage of wild-type (WT) and ifnar1 -/- iBMDMs with and without associated Kp52145 positive for CD206 1, 3 or 5 h post infection. Kp52145 was tagged with mCherry.Percentage of wild-type (WT) and ifnar1 -/- iBMDMs with and without associated Kp52145 positive for MHCII 1, 3 or 5 h post infection. Kp52145 was tagged with mCherry.Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf3 -/- for 60 or 120 min.Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or irf31 -/- for 60 or 120 min. 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. ****P = 0.0001; ***P = 0.001; **P = 0.01; *P = 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.

Fig 4: Klebsiella pneumoniae-induced M(Kp) polarisation is dependent on IL10 Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or tlr4 -/- for 60 or 120 min.Immunoblot analysis of phospho-CREB (pCREB) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or myd88 -/- for 60 or 120 min. il0 mRNA levels were assessed by qPCR in iBMDMs transfected with All Stars siRNA control (AS), or CREB siRNA (CREBsi) non-infected (ni) or infected with Kp52145 for 3 h.Immunoblot analysis of phospho-STAT6 (pSTAT6) and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10 -/- for 60 or 120 min. arg1 mRNA levels were assessed by qPCR in wild-type (WT), il10 -/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.Immunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type (WT), or il10 -/- for 60 or 120 min. pparg mRNA levels were assessed by qPCR in wild-type (WT), il10 -/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h. fizz1 mRNA levels were assessed by qPCR in wild-type (WT), il10 -/- non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.Kp52145 adhesion to wild-type (WT) and il10 -/- iBMDMs. Cells were infected with Kp52145 for 30 min, washed, cell lysed with saponin, and bacteria quantified after serial dilution followed by plating on LB agar plates.Phagocytosis of Kp52145 by wild-type (WT) and il10 -/- iBMDMs. Cells were infected for 30 min, wells were washed, and it was added medium containing gentamicin (100 µg/ml) to kill extracellular bacteria. After 30 min, cells were washed, cell lysed with saponin and bacteria quantified after serial dilution followed by plating on LB agar plates.Kp52145 intracellular survival in wild-type (W)T and il10 -/- 5 h after addition of gentamycin (30 min of contact). Results are expressed as % of survival (CFUs at 5 h versus 30 min in stat6 -/- cells normalised to the results obtained in wild-type macrophages set to 100%).Immunofluorescence confocal microscopy of the co-localisation of Kp52145 harbouring pFPV25.1Cm, and cresyl violet dye in wild-type (WT) and il10 -/- macrophages. The images were taken 90 min post infection. Images are representative of duplicate coverslips of three independent experiments.Percentage of Kp52145 harbouring pFPV25.1Cm co-localisation with cresyl violet over a time course in wild-type (WT) and il10 -/- macrophages. Values are given as mean percentage of Kp52145 co-localising with the marker ± SEM. The number of infected cells counted per time in three independent experiments are indicated in the figure. 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. In panels (C, P, Q and R) unpaired t test was used to determine statistical significance. In all the other panels, 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.

Fig 5: Klebsiella pneumoniae-induced M(Kp) polarisation is STAT6 dependent AImmunoblot analysis of the levels of the indicated proteins in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.B klf4 mRNA levels were assessed by qPCR in wild-type iBMDMs infected with Kp52145 for 1, 3 or 5 h.CImmunoblot analysis of Klf4 and tubulin levels in lysates from non-infected (ni) and infected wild-type iBMDMs for 60 or 120 min.D–FAnalysis by flow cytometry of the levels of M(Kp) markers expressed by IMs from PBS-mock-infected mice (black dots), and by IMs from infected mice (blue dots) with and without associated Kp52145. (D) Percentage of positive cells for Arg1. (E) Percentage of positive cells for Fizz1. (F) Percentage of positive cells for iNOS.G arg1 mRNA levels were assessed by qPCR in wild-type and stat6 -/- iBMDMs non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h.HImmunoblot analysis of Arg1 and tubulin levels in lysates from non-infected (ni) and infected wild-type and stat6 -/- iBMDMs for 60 or 120 min.I il10 mRNA levels were assessed by qPCR in wild-type (WT) and stat6 -/- iBMDMs non-infected (ni) or infected with Kp52145 for 1, 3 or 5 h. Data information: For all infections, after 1 h contact, medium replaced with medium containing gentamycin (100 µg/ml) to kill extracellular bacteria. qPCR and flow cytometry values are presented as the mean ± SEM of three independent experiments measured in duplicate. Images are representative of three independent experiments. ****P = 0.0001; ***P = 0.001; **P = 0.01; *P = 0.05; ns, P > 0.05 for the indicated comparisons using one way-ANOVA with Bonferroni contrast for multiple comparisons test.