Figure 6.

KDM6B demethylase activity is necessary for chronic Salmonella infection and M2 macrophage polarization. [A] Schematic representation of Salmonella chronic infection model (using attenuated aroA mutant SL3261 strain was used) along with GSKJ4 treatment. Panel [B] and [C] represent SL CFU obtained from spleen and MLN 30 days post chronic SL infection (dpi) in vehicle and GSKJ4 treated animals.[D] Representative of M2 macrophage population estimated using flow cytometry analysis of CD206+CD301+ double positive cell population on CD45.2+CD11b+Ly6G- cell population in spleen following 30 dpi. (Fig D is representative of FACS analysis of splenocytes of one of the mice from each group). CD206+CD301+ double positive cell frequency in spleen [E] and MLN [F] 30 days post chronic SL infection (dpi) in vehicle and GSKJ4 treated animals (% CD206+CD301+ population in CD45.2+CD11b+Ly6G-) (Gating strategy used is shown in Fig S4; n = 3 mice per group). Mean fluorescence intensity of CD301 expression on CD45.2+CD11b+Ly6G- population in spleen. [H] Flow cytometry analysis of CD301 population in GSKJ4 (5 µM) treated or KDM6B knock down BMDM at 24h p.i.[I] Graphical representation of CD301 population upon KDM6B knockdown or GSKJ4 treatment in Salmonella infected BMDMs in presence of IL4 (20 ng/ml). Salmonella infected or KDM6B perturbed BMDMs using GSKJ4 or KDM6B siRNA were anlyazed for M2 macrophage markers [J] and fatty oxidation pathway genes [K] using qRT-PCR array 24 h p.i. Statistical significance was analyzed using Mann Whitney U test (b-c), one way Anova analysis with post Tukey test (E,F,I) and student t- test (j-k). (‘***’- p-value <.001; ‘**’- p-value <.01, ‘*’ p-value <.05)