RAS-p110α signalling in macrophages is required for effective inflammatory response and resolution of inflammation

Alejandro Rosell
Agata A. Krygowska
Marta Alcón Pérez
Mathieu-Benoit Voisin
Juan de Paz
Héctor Sanz-Fraile
Vinothini Rajeeve
Alberto Berral-González
Cristina Cuesta
Ottilie Swinyard
Enrique Gabandé-Rodriguez
Julian Downward
Jordi Alcaraz
Javier De Las Rivas
Pedro Cutillas
Esther Castellano author has email address

Tumour-Stroma Signalling Lab., Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
Centre for Cancer and Inflammation, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom
Centre for Microvascular Research, William Harvey Research Institute, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, United Kingdom
Unit of Biophysics and Bioengineering, Department of Biomedicine, School of Medicine and Health Sciences, Universitat de Barcelona, Barcelona, Spain
Centre for Cancer Genomics and Computational Biology, Cell Signalling and Proteomics Laboratory, Barts Cancer Institute, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, United Kingdom
Bioinformatics and Functional Genomics, Centro de Investigación del Cáncer, Instituto de Biología Molecular y Celular del Cáncer, Consejo Superior de Investigaciones Científicas (CSIC)-Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
Oncogene Biology Laboratory, Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK
Institute for Bioengineering of Catalonia (IBEC), The Barcelona Institute for Science and Technology (BIST), Barcelona 08028, Spain

https://doi.org/10.7554/eLife.94590.1

Open access
Copyright information

Figures and data

Pik3ca^RBD/- mice have impaired responses to inflammatory insults.
(A) Western blotting showing activation of Akt and ERK in Pik3ca^WT/- and Pik3ca^RBD/- BMDMs activated towards a pro-inflammatory state with LPS and IFN-࿎ at the indicated time points; (B)) Western blotting showing activation of NF-kB (p65) in Pik3ca^WT/- and Pik3ca^RBD/- BMDMs activated towards a pro-inflammatory state with LPS and IFN-࿎ at the indicated time points; (C) Pik3ca^WT/- and Pik3ca^RBD/- mice were injected with zymosan or PBS in the back-hind paws and inflammation was measured and plotted over time, Pik3ca^WT/- PBS n = 6; Pik3ca^RBD/- PBS n = 5; Pik3ca^WT/- Zymosan n = 4; Pik3ca^RBD/- Zymosan n = 4; Error bars indicate mean ± SEM. Significance using 2-way ANOVA test: **p < 0.01 (D and E) Representative H&E images of the inflamed area of Pik3ca^WT/- and Pik3ca^RBD/- paws injected with zymosan at the indicated times; (F) Representative images of cellularity present in the inflamed abscess of Pik3ca^WT/- and Pik3ca^RBD/- paws injected with zymosan at the indicated times. Blue arrows indicate apoptotic polymorphonuclear cells, yellow arrows indicate fibrin deposition, green arrows indicate macrophages and orange arrows indicate activated fibroblasts; (G) Representative images of macrophages (CD68 positive cells) present in the inflamed abscess of Pik3ca^WT/- and Pik3ca^RBD/- paws injected with zymosan; (H) Quantification of macrophages (CD68 positive cells) present in the inflamed abscess of Pik3ca^WT/- and Pik3ca^RBD/- paws injected with zymosan; (I) Representative H&E images of the inflamed area and cellularity of Pik3ca^WT/WT paws and Pik3ca^WT/WT paws treated with BYL-719 injected with zymosan. Blue arrows indicate apoptotic polymorphonuclear cells, yellow arrows indicate fibrin deposition, green arrows indicate macrophages; (J) Representative images and quantification of macrophages (CD68 positive cells) present in the inflamed abscess of Pik3ca^WT/WT paws and Pik3ca^WT/WT paws treated with BYL-719 injected with zymosan; (K) Quantification of macrophages (CD68 positive cells) present in the inflamed abscess of Pik3ca^WT/WT paws and Pik3ca^WT/WT paws treated with BYL-719 injected with zymosan. Error bars indicate mean ± SEM. Significance using Student’s t test: **p < 0.01.

Disruption of RAS-p110α interaction decreases the number of inflammatory monocytes in blood and spleen.
Twelve-week-old mice were treated with tamoxifen and after four weeks, flow cytometry analysis was performed to determine (A) inflammatory monocytes in circulating blood; (B) alternatively activated monocytes in circulating blood; (C) inflammatory monocytes in spleen; (D) classically activated monocytes in spleen; (E) macrophages in spleen; (F) macrophages in spleen’s white pulp; (G) macrophages in spleen’s red pulp. Data are presented as percentage of positive cells for the indicated markers. Black dots represent data from Pik3ca^W/- mice and red dots represent data from Pik3ca^RBD/- mice. Each dot represents an individual mouse. Error bars indicate mean ± SEM. Significance using Student’s t test: * p < 0.05; **p < 0.01.

Disruption of RAS-p110α interaction impairs transendothelial extravasation to sites of inflammation.
(A) Graph indicating the quantification of Pik3ca^RBD/- and Pik3ca^RBD/- BMDMs passing through 8 μm membrane pore transwells for 24 hours. Membrane attached macrophages on the lower part of the transwell were stained with crystal violet and quantified. Each dot represents an independent experiment. Error bars indicate mean ± SEM; (B) Random migration of Pik3ca^WT/, Pik3ca^RBD/- and Pik3ca^WT/WTBMDMs treated with BYL719 (500 ng/ml) was analysed by time-lapse video microscopy and cell tracing in the presence or absence of LPS (100 ng/ml) and IFN-࿎ (20 ng/ml); (C) Schematic representation of myeloid chimera generation strategy; (D) Graph showing level of bone marrow reconstitution with Pik3ca^RBD/- and Pik3ca^WT/- myeloid lineage; (E) Graph quantifying the number of neutrophils in the blood of chimera mice treated with anti-GR1 (25μg/mouse/day); (F-H) Intra vital imaging quantification of the number of: (F) rolling monocytes per 5 minutes; (G) adherent monocytes per 500 μm of vessel segment; (H) extravasated monocytes per mm² of tissue. Each dot represents an individual mouse. (I) Chimera Pik3ca^W/- and Pik3ca^RBD/- mice were injected with zymosan or PBS in the back-hind paws and inflammation was measured and plotted over time, Pik3ca^WT/- PBS n = 5; Pik3ca^RBD/- PBS n = 5; Pik3ca^WT/- Zymosan n = 5; Pik3ca^RBD/- Zymosan n = 5.
Statistical significance was obtained using Mann-Whitney test (A-H) or 2-way ANOVA test (I): n.s. non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Disruption of RAS-p110α signalling impairs BMDM ability to remodel their cytoskeleton in response to inflammatory cues.
(A) Representative images of Pik3ca^WT/- and Pik3ca^RBD/- BMDMs unstimulated or activated with LPS+ IFN-࿎; (B) violin plot quantifying spread area. IF images of Pik3ca^WT/- and Pik3ca^RBD/- BMDMs co-stained with phalloidin and DAPI were used to analyse spread area. Results are represented as a violin plot. Three independent biological replicates were analysed (n ≥ 250 total cells); (C) Quantification of cell circularity of Pik3ca^WT/- and Pik3ca^RBD/- BMDMs unstimulated or activated with LPS+ IFN-࿎. Quantification was performed using the same images from panel (A); (D) Representative 3D projections of Pik3ca^WT/- and Pik3ca^RBD/- BMDMs unstimulated or activated with LPS+IFN-࿎ and violin plot showing quantification of the corresponding cell height. 3D projections and cell height were analysed using same images used in panel (A). (E) Violin plot representing F-actin pool in Pik3ca^WT/- and Pik3ca^RBD/- BMDMs unstimulated or activated with LPS+ IFN-࿎. Three independent biological replicates were analysed (n ≥ 250 total cells); (F) Violin plot representing G-actin pool (measured by DNAse staining) in Pik3ca^WT/- and Pik3ca^RBD/- BMDMs unstimulated or activated with LPS+ IFN-࿎. Three independent biological replicates were analysed (n > 250 total cells); (G) Pik3ca^WT/- and Pik3ca^RBD/- BMDMs were activated with LPS+ IFN-࿎ and western blots were performed to determine expression levels of the indicated proteins.
Statistical significance was obtained using Mann-Whitney test: n.s. non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Secretome analysis of Pik3ca^RBD/- BMDMs suggested a defect in complement activation and lysosomal function.
(A and B) Volcano plots of secretome analysis from Pik3ca^WT/- and Pik3ca^RBD/- BMDMs in non-stimulated conditions (A) or phagocytosing apoptotic cells (B). The x-axis shows the log2 FC of each identified protein and the y-axis the corresponding -log10 P value. Statistically significant peptides with FC >2 and p-value < 0.05 are in blue; peptides that do not pass this threshold are in grey; peptides with FC > 2 but p-value ≥ 0.05 are in green; (C) Venn diagram showing the overlap of Pik3ca^RBD/- vs Pik3ca^WT/- BMDMs differentially expressed proteins at rest and during phagocytosis of apoptotic cells. Proteins displayed in the blue square represents peptides differentially expressed in resting BMDMs, the brown square represents peptides differentially expressed during phagocytosis, and the peptides differentially expressed in both conditions are displayed in purple; (D and E) Network analysis of significantly expressed proteins identified in the secretome analysis of Pik3ca^RBD/- vs Pik3ca^WT/- BMDMs in steady state conditions (D) or during phagocytosis of apoptotic cells (E). The nodes represent individual proteins, and the edges represent known interactions between proteins, either co-expression (purple) or physical interaction (thick pink). The size of each node reflects the significance of differential expression. Proteins in green have been implicated in lysosomal function, while proteins in orange are members of the complement cascade.

Disruption of RAS-p110α activation in BMDMs leads to abnormal lysosomal function
(A) Representative IF images and quantification analysis of lamp intensity in Pik3ca^WT/- and Pik3ca^RBD/- BMDMs in steady state conditions and during phagocytosis of apoptotic LKR10 cells. Three independent biological replicates were analysed (n ≥ 250 total cells). Error bars indicate mean ± SEM; (B) Quantification analysis of lysosomal function in Pik3ca^WT/-, Pik3ca^RBD/- and Pik3ca^WT/WT BMDMs treated with BYL719 BMDMs unstimulated or activated with LPS + IFN-࿎ using Lysotracker staining. Three independent biological replicates were analysed (n ≥ 250 total cells); (C) Violin plot displaying lysosome pH acidity in unstimulated and LPS+IFN-࿎ stimulated Pik3ca^W/- (black), Pik3ca^RBD/- (red) and Pik3ca^WT/WT BMDMs treated with BYL719 (Blue) BMDMs, as determined by Lysosensor staining. Three independent biological replicates were analysed (n ≥ 250 total cells); (D) Pik3ca^WT/- and Pik3ca^RBD/- BMDMs were activated with LPS (100 ng/ml) and IFN-࿎ (20 ng/ml) and expression and activation of Cathepsin B and D were blotted; (E) Quantification of phagocytosed apoptotic cells in Pik3ca^WT/-, Pik3ca^RBD/- and Pik3ca^WT/WT BMDMs treated with BYL719 BMDMs. Apoptotic cells were labelled with a red tracker and allowed to be phagocytosed by control, Pik3ca^RBD/- and Pik3ca^WT/WT BMDMs treated with BYL719 for 16 hours, measuring the amount of cell tracker that was internalized at different time points. Each dot of the graph indicates a different independent experiment. Error bars indicate mean ± SEM; (F) Graph displaying the ability of Pik3ca^WT/- and Pik3ca^RBD/- BMDMs to degrade GFP-labelled (pH sensitive) and red tracker-labelled (pH insensitive) apoptotic cells. Three independent biological replicates were analysed. Error bars indicate mean ± SEM; (G) Representative image and quantification of Cathepsin D expression in the inflammatory abscess of Pik3ca^WT/- and Pik3ca^RBD/- paws injected with zymosan; (H) Representative image and quantification of Cathepsin D expression in the inflammatory abscess of paws from control and Pik3ca^WT/WT mice treated with BYL719 injected with zymosan.
Statistical significance was obtained using Mann-Whitney test: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Pik3ca^RBD/- mice have more extended inflamed tissue after zymosan injection
(A) Schematic representation of the mouse model used in which the interaction of p110α with RAS was disrupted by the introduction of two-point mutations, T208D and K227A, into the endogenous Pik3ca gene (Pik3ca^RBD). Wild-type (Pik3ca^WT) and Pik3ca^RBD mice were bred with mice containing a floxed Pik3ca allele and a mouse carrying a conditional Cre recombinase (Cre-ERT2) allele targeted to the ubiquitously expressed Rosa26 locus; (B) Representative PCR showing efficiency of floxed allele removal after treatment with 4-hydroxytamoxifen for 48 hours; (C) Pik3ca^WT/Flox and Pik3ca^RBD/Flox mice were injected with zymosan or PBS in the back-hind paws and inflammation was measured and plotted over time, Pik3ca^WT/Flox n = 5; Pik3ca^RBD/Flox PBS n = 5; Pik3ca^WT/Flox Zymosan n = 5; Pik3ca^RBD/Flox Zymosan n = 5; Error bars indicate mean ± SEM. Significance using 2-way ANOVA test: n.s. non-significant (D) Blood from the same mice was obtained by cardiac puncture prior culling and blood sedimentation was measured. The clear layer formed at the top was used for data quantification. Black dots represent data from Pik3ca^WT/- mice and red dots represent data from Pik3ca^RBD/~ mice. Each dot represents an individual mouse. Each dot represents an individual mouse. Error bars indicate mean ± SEM. Significance using Student’s t test: * p < 0.05; **p < 0.01. Pik3ca^WT/- PBS n = 6; Pik3ca^RBD/' PBS n = 5; Pik3ca^WT/- Zymosan n = 4; Pik3ca^RBD/' Zymosan n = 4; (E) Representative images showing zymosan-induced inflammatory area in Pik3ca^WT/WT mice and Pik3ca^WT/WT mice treated with BYL-719.

Disruption of RAS-p110α interaction does not affect the number of B-cells or T-cells in blood or spleen.
Twelve-week-old mice were treated with tamoxifen and after four weeks, flow cytometry analysis was performed to determine abundancy of different immune populations. (A) Granulocytes in circulating blood; (B) CD3, CD4+ and CD8+ T-cells in circulating blood; (C) B-cells in circulating blood; (D) Granulocytes in spleen; (E) B-cells in spleen; (F) T-cells in spleen; (G) Spleen weight compared to full body; (H) Schematic representation of myeloid precursors differentiation (CMP-common myeloid progenitor; GMP-Granulocyte-monocyte progenitor; MEP-Megakaryocyte-erythroid progenitor); (I) Graph showing percentage of bone marrow cells progenitor cells (have not acquire any lineage marker, Lin^-); (J) Graph showing percentage of CMP cells within progenitor cells; (K) Graph showing percentage of MEP cells within progenitor cells; (L) Graph showing percentage of GMP cells within progenitor cells; (M) Graph depicting % of differentiated macrophages obtained in culture from bone marrow precursors in the presence of m-CSF. Black dots represent data from Pik3ca^WT/- mice and red dots represent data from Pik3ca^RBD/~ mice. Each dot represents an individual mouse. Error bars indicate mean ± SEM. Significance using Student’s t test: n.s. non-significant.

Pik3ca^RBD/- monocytes do not extravasate through the endothelium
(A) Graph showing levels of neutrophil reconstitution in chimera’s bone marrow. Blood from chimera mice was collected and GFP in granulocytes was analysed by flow cytometry to determine engraftment percentage. (B) Graph quantifying the number of monocytes in the blood of chimera mice treated with anti-GR1 (25μg/mouse/day) for 3 days. (C) Graph showing levels of expression of CCR2 in Pik3ca^WT/~ and Pik3ca^RBD/~ BMDMs detected by flow cytometry. Error bars indicate mean ± SEM. Significance using Mann-Whitney test: n.s. no-significant

Disruption of RAS-p110α interaction alters the secretome of BMDMs
Pik3ca^RBD/ and Pik3ca^RBD/ BMDMs were stimulated with LPS+IFN-y and expression of (A) CD80; (B) CD86; and (C) MHCII were analysed by flow cytometry (Pik3ca^RBD/ n = 5; Pik3ca^WT/ n = 3). Error bars indicate mean ± SEM. Significance using Mann-Whitney test: n.s. no-significant. (D) WB showing induction of apoptosis in LKR10 cell line treated with cisplatin for 16 hours; (E) Dendrogram showing hierarchical clustering of peptides found in the secretome analysis of Pik3ca^RBD/ and Pik3ca^RBD/ BMDMs in steady state conditions; (F) Dendrogram showing hierarchical clustering of peptides found in the secretome analysis of Pik3ca^RBD/ and Pik3ca^RBD/ BMDMs phagocytosing apoptotic cells.

Differentially expressed peptides identified in Pik3ca^RBD/- vs Pik3ca^WT/- secretome of BMDMs under steady state conditions

Differentially expressed peptides identified in Pik3ca^RBD/- vs Pik3ca^WT/- secretome of BMDMs under steady state conditions

Differentially expressed peptides identified in Pik3ca^RBD/- vs Pik3ca^WT/- secretome of BMDMs phagocytosing apoptotic cells

Differentially expressed peptides identified in Pik3ca^RBD/- vs Pik3ca^WT/- secretome of BMDMs phagocytosing apoptotic cells

Sign up for email alerts