Fig 2: PGE2 impacts macrophage differentiation, but not macrophage polarization. (A) To determine the effect of PGE2 signaling on human macrophages, CD14+ monocytes were differentiated for 6 days with M‐CSF before polarizing them with LPS/IFN‐γ (M1), IL‐4/IL‐13 (M2), or PGE2. After 2 days of polarization, cells were analyzed for marker expression or used for functional assays such as T cell suppression, migration, or phagocytosis assays. (B) The marker expressions of polarized macrophages are depicted as bar graphs showing the mean fluorescent intensity (MFI) of CD206, CD163, MerTK, CD14, CD80, PD‐L1, and HLA‐DR. Bar graphs show the mean ± SEM, and each dot represents an individual donor (n ≥ 5) combined from two independent experiments. p‐values were calculated with one‐way ANOVA with Tukey multiple comparison correction or Kruskal–Wallis test with Dunn's multiple comparison correction. Additionally, histograms display the expression levels of each of those markers for one representative donor. (C) Monocytes nondifferentiated (/) or differentiated with M‐CSF, or M‐CSF + PGE2 for 6 days. MFI of CD206, CD163, MerTK, HLA‐DR, and CD11b is shown as bar graphs with the mean ± SEM, and each dot represents an individual donor (n = 4) from two independent experiments. p‐values were calculated using one‐way ANOVA with Tukey multiple comparison correction. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Fig 3: ACK1 and BRK kinase deficiency disrupts the anti-inflammatory response driven by apoptotic cells in macrophages.(A) Western blot analysis for AKT phosphorylation by ACK1 and BRK. Cell lysates from 293T cells were incubated with anti-AKT. Immunoprecipitated proteins were probed with anti-phosphotyrosine and anti-AKT antibodies. (B) Western blot analysis for STAT3 phosphorylation by ACK1 and BRK. Lysates from 293T cells coexpressing STAT3 and Flag-tagged WT or mutant forms (A156T and K161Q) of ACK1 or mutant forms (G257A and G321R) of BRK were probed with anti-phospho-STAT3 (Tyr705), anti-STAT3 and anti-Flag antibodies. For analysis of BRK, cells were treated with 100 ng/ml EGF for 10 min. (C) RAC activation by WT ACK1 and BRK. Cell lysates from 293T cells expressing WT or mutant forms of ACK1 (left) and lysates from 293T cells expressing WT or mutant forms of BRK (right) were incubated with GST-PAK CRIB sepharose beads, and the level of RAC1 GTP was determined by immunoblotting with anti-Rac1 antibody. Lysates were also probed with anti-Rac1, anti-FLAG and anti-tubulin antibody. For analysis of BRK, 293T cells were cotransfected with CAS and stimulated with 100 ng/ml EGF for 10 min. (D) MERTK increases kinase activity of BRK and ACK1. IP kinase assay. ACK1 (left) was immunoprecipitated from 293T cells co-transfected with Flag-tagged ACK1 WT, ACK1 A156T, or ACK1 K161Q and MERTK with anti-Flag Ab. Immunoprecipitated proteins were used in duplicate in vitro for kinase reactions with WASP synthetic peptide and results represented as pmol phosphate transferred. BRK (right) was immunoprecipitated as above from 293T cells co-transfected with Flag-tagged BRK WT or mutants and MERTK with anti-Flag Ab. Kinase reactions was performed with peptide AEEEIYGEFEAKKKG, and represented as above. p-Values were calculated using an Anova test (Tukey’s multiple comparison test). (E) Regulation of inflammatory response. Significant normalized enrichment scores (NES) for GO ‘positive regulation of acute inflammation’ gene set, GO ‘negative regulation of inflammatory response’ gene set, and GO ‘AKT_UP.V1_UP’ gene set in WT and mutant macrophages, and WT treated with AIM100 (2 µM) or Cpd4f (0.5 µM), exposed to apoptotic cells, with three replicates per experimental condition. Significant enrichment (p-value <0.05 and FDR (q-value) <0.25) are calculated as reported in Materials and methods. (F) Table of the top 10 differentially regulated genes by apoptotic cells in WT macrophages are not differentially expressed in mutant macrophages and WT macrophages treated with AIM100 or Cpd4f (treated as in E). Numbers indicate FDR (q-value). Known target genes of STAT3 and AKT are labeled in blue and red respectively (G) TNF mRNA production by WT macrophages treated with AIM100 (2 µM) 4 hr after exposure to apoptotic cells. n=6, from two independent experiments. (H,I) TNF and IL1β production by macrophages, as measured by ELISA on media collected from mutant and isogenic WT macrophages (C12.1) incubated with mouse apoptotic thymocytes for 90 min, then stimulated with LPS (1 ng/ml) for 18 hr. n≥4, from ≥2 independent experiments. p-Values in H were calculated by Wilcoxon matched-pairs signed rank tests for data that is not normally distributed, while p-values in G and I were calculated using an Anova test with Tukey’s correction for multiple comparisons. Figure 4—source data 1.Figure 4D: Immunoprecipitation (IP) kinase activity assay of WT and mutant ACK1 and BRK kinases in HEK293T cells in the presence of MERTK and/or GAS6. Figure 4—source data 2.Figure 4E: GSEA of control, inhibitor treated, and mutant iPSC-derived macrophages in the presence or absence of apoptotic cells. Figure 4—source data 3.Figure 4G: TNF mRNA production by WT AIM100 treated macrophages 4 hr after exposure to apoptotic cells. Figure 4—source data 4.Figure 4H: TNF protein production by WT and mutant iPSC-derived macrophages in the presence or absence of LPS and/or apoptotic cells. Figure 4—source data 5.Figure 4I: IL1b protein production by WT and mutant iPSC-derived macrophages in the presence of LPS and/or apoptotic cells. Figure 4—source data 6.Uncropped and labeled gels for Figure 4. Figure 4—source data 7.Raw unedited gels for Figure 4.

Fig 4: Generation and characterization of control and patient derived iPSCs and iPSC-macrophages.(A) Schematic representation of the process of reprograming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). See Materials and methods for details. (B) Sanger sequencing of reprogrammed iPSC lines from unrelated wild type (WT) control (C12), familial controls (1-II-3 ACK1WT/K161Q and 2-II-3 BRKWT/G321R), and SLE patients (1-III-1 ACK1A156T/K161Q and 2-III-3 BRKG257A/G321R). Gray bars indicate positions of nucleotide substitution. (C) Nucleic acid and predicted amino acid sequences for both gene copies of TNK2(AKC1) or PTK6(BRK) in iPSC lines from C12 and SLE families. Variations from WT are indicated in red. (D) Karyotypes of iPSC lines from C12 and SLE families. Chromosome analysis was performed on a minimum of 20 DAPI-banded metaphases. (E) Flow cytometry analysis of surface receptor expression on iPSC-derived macrophages from C12, familial controls, and SLE patients iPSC lines. Histograms show fluorescence intensity for indicated antibodies (red) and FMO controls (grey). (F) Bar plots show percent viability based on DAPI staining of macrophages derived from C12, familial controls, and SLE patients iPSC lines. n≥12, from three independent experiments. (G) RT-QPCR analysis of mRNA for BRK, ACK1, TIM4, and MERTK. mRNA expression was normalized to GAPDH (2-ΔCt; see Materials and methods). n≥3, from one to two independent experiments. p-Values were obtained using an Anova test with Tukey’s correction for multiple comparisons.