In vivo single cell transcriptomics reveal K. pneumoniae skews lung macrophages to promote infection (2022)
Figure 8

Figure 8. K. pneumoniae induces M(Kp) polarization in human macrophages.

A. 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.

B. 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.

C. 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.

D. 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.

E. 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

F. 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.

G. 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.

H. 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.

I. 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.

J. 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.

K. 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.

L. 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.

M. 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.

N. 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.

O. 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-ΔwcaK2, for 60 or 120 min.

P. Percentage of PMA-treated THP-1 macrophages with and without associated Kp52145 or 52145-ΔwcaK2 positive for Arg1 5 h post infection. Bacteria were tagged with mCherry.

Q. Percentage of PMA-treated THP-1 macrophages with and without associated Kp52145 or 52145-ΔwcaK2 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.

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