In vivo single cell transcriptomics reveal K. pneumoniae skews lung macrophages to promote infection (2022)
Figure 6

Figure 6. K. pneumoniae-induced M(Kp) polarisation is dependent on IL10.

A. 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.

B. 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.

C. 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.

D. 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.

E. 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.

F. 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.

G. 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.

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.

I. 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.

J. 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.

K. 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 normalized to the results obtained in wild-type macrophages set to 100%).

L. Immunofluorescence confocal microscopy of the colocalization 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.

M. Percentage of Kp52145 harbouring pFPV25.1Cm co-localization with cresyl violet over a time course in wild-type (WT) and il10-/- macrophages. Values are given as mean percentage of Kp52145 co-localizing 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.