Figure 1.
Macrophages express RGS10 and its expression is regulated by LPS.
A) RGS mRNA levels in mouse BMDMs were assessed by quantitative PCR. Cycle threshold (Ct) values reveal that RGS10 is the most abundantly expressed gene. B) Peripheral blood monocytes and splenic macrophages express RGS10 at levels detectable by intracellular flow cytometry staining. Intracellular RGS10 expression in CD11b+CD14+CD45+ monocytes (a) and splenic macrophages (b) was measured by flow cytometry in Rgs10-/- (shaded) and WT (unshaded) mice. C) Representative western blot data and D) quantitative analysis of expression of RGS10 protein levels in bone marrow-derived macrophages (BMDM) upon LPS (100 ng/mL) treatment. The results represent the mean +/- SEM of three independent experiments. Student t-test; **, p< 0.01 or ***, p <0.001.
Figure 2.
Rgs10-/- macrophages are hyper-responsive to LPS.
A) Levels of pro-inflammatory cytokines and chemokines (TNF, IL-12 p40, IL-6, IL-1b, KC, IL-10) produced by wild type and Rgs10-/- BMDMs. Data are from one experiment representative of three independent experiments. Cells were plated and treated with LPS (100 ng/mL) for 24 hours as indicated in Materials and Methods. Conditioned medium was analyzed by Multiplexed immunoassay ELISA system (Meso Scale Discovery). Statistical analysis is described in Materials and Methods. *, p < 0.05;***p <0.001 compared within the group. #, p < 0.05; ##, p< 0.01; ###p <0.001 compared between the groups. B) Western blot analysis data of phospho-p65RelA and p65RelA expression in Rgs10-/- BMDMs after LPS (100 ng/mL) stimulation as indicated time. Data are from one experiment representative of three independent experiments. C) Conditioned media from BMDMs treated with LPS (100 ng/mL) for 24 hours were collected. MN9D neuroblastoma cells were treated with BMDM conditioned media for 48 hours. Cytotoxicity was measured by the MTS assay as described in Materials and Methods section. Values shown represent group means (n=4) ± S.E.M from one experiment representative of two independent experiments. *** denotes significant differences between vehicle and LPS within the group at p < 0.001 respectively. ## denotes significant differences between groups at ###p < 0.001.
Figure 3.
Rgs10-/- bone marrow derived macrophages (BMDMs) exhibit attenuated M2 activation phenotype.
A) mRNA expression of YM1, Fizz1 and Arg1 mRNA from BMDMs treated with IL-4 (10 ng/mL) for 4 hours. Data are from one experiment representative of three independent experiments. B) Western blot analysis of YM1, Arg 1 and RGS10 proten expression in WT and Rgs10-/- BMDMs after IL-4 (10 ng/mL) treatment for the indicated times. C) IL-4R expression (with isotype control antibody in black) in CD45+CD11b+CD14+ BMDMs from WT (blue) or Rgs10-/- (red) mice. D) mRNA levels of YM1 and FIZZ1 in BMDMs upon LPS stimulation after IL-4 (10 ng/mL) priming for 48hours. Statistical analysis is described in Materials and Methods. **, p < 0.01;***p <0.001 compared within the group. ##, p< 0.01; ###p <0.001 compared between the groups. E) BMDMs were treated with LPS for the indicated times after IL-4 (10 ng/mL) priming for 48hours. Western blot analysis of YM1 protein in BMDMs from WT or Rgs10-/- mice. Data are from one experiment representative of three independent experiments.
Figure 4.
Rgs10-/- peritoneal macrophages display exaggerated classical activation and impaired alternative activation phenotypes.
A) Production of cytokines and chemokines (TNF, IL-12 p40, IL-6, IL-1b, KC, IL-10) by WT and Rgs10-/- peritoneal macrophages. Peritoneal macrophages were isolated and treated with LPS (100 ng/mL) for 24 hours. Cultured medium was analyzed by Multiplexed immunoassay ELISA system (Meso Scale Discovery). Data are from one experiment representative of three independent experiments. B) Peritoneal macrophages were treated for 48 hours with IL-4, IL-13 or IFNγ (10 ng/mL) priming and challenged with LPS (100 ng/mL) for 24 hours. Levels of YM1, Fizz1 and Arg1 were determined by quantitative real-time PCR. C) Nitrite release was measured using the Griess reagent assay. Statistical analysis is described in Materials and Methods. **, p < 0.01;***p <0.001 compared within the group. ##, p< 0.01; ###p <0.001 compared between the groups.
Figure 5.
Rgs10-/- macrophages exhibit blunted phagocytic activity but normal chemotaxis.
A) Phagocytic activity upon exposure to Escherichia coli (E. coli) particles is reduced in Rgs10-/- macrophages compared to WT macrophages. BMDMs were primed with IL-4 (10 ng/mL) for 48 hours. Each experimental condition was performed in quadruplicate. B) Chemotaxis of Rgs10-/- macrophages induced by LPS (1 µg/mL) or FBS (20%) is indistinguishable from that of WT macrophages. Data are from one experiment representative of three independent experiments. Cells were treated as described in Materials and Methods. Statistical analysis is described in Materials and Methods. #, p<0.05; ##, p< 0.01; ###p <0.001 compared between the groups.