RIP2 promotes FcγR-mediated reactive oxygen species production

Abstract

Receptor-interacting protein 2 (RIP2) is a kinase that mediates signaling downstream of the bacterial peptidoglycan sensors NOD1 and NOD2. Genetic loss or pharmaceutical inhibition of RIP2 has been shown to be beneficial in multiple inflammatory disease models with the effects largely attributed to reducing proinflammatory signaling downstream of peptidoglycan recognition. However, given the widespread expression of this kinase and its reported interactions with numerous other proteins, it is possible that RIP2 may also function in roles outside of peptidoglycan sensing. In this work, we show that RIP2 undergoes tyrosine phosphorylation and activation in response to engagement of the Fc γ receptor (FcγR). Using bone marrow-derived macrophages from WT and RIP2-KO mice, we show that loss of RIP2 leads to deficient FcγR signaling and reactive oxygen species (ROS) production upon FcγR cross-linking without affecting cytokine secretion, phagocytosis, or nitrate/nitrite production. The FcγR-induced ROS response was still dependent on NOD2, as macrophages deficient in this receptor showed similar defects. Mechanistically, we found that different members of the Src family kinases (SFKs) can promote RIP2 tyrosine phosphorylation and activation. Altogether, our findings suggest that RIP2 is functionally important in pathways outside of bacterial peptidoglycan sensing and that involvement in such pathways may depend on the actions of SFKs. These findings will have important implications for future therapies designed to target this kinase.

Keywords: Fc receptor; Fc-γ receptor; Src; inflammation; reactive oxygen species (ROS); receptor interacting protein 2 (RIP2); signal transduction.

Figure 1.

RIP2 is tyrosine-phosphorylated and activated upon FcγR cross-linking. A, sequence surrounding the Tyr-474 autophosphorylation site on RIP2, showing that it fulfils the criteria for a Src-family kinase SH2-binding motif. RAW 264.7 macrophages (B) and WT BMDMs (C) were stimulated with murine anti-BSA IgG₁ + BSA for the indicated times. RIP2 was immunoprecipitated, Western blotting was performed, and IPs were immunoblotted using an anti-phosphotyrosine antibody. Total cell lysates were immunoblotted with the indicated antibodies to assess activation of downstream signaling cascades. IVK assays were performed using RIP2 immunoprecipitated from FcγR-stimulated RAW 264.7 cells (D) and WT BMDMs (E) using tyrosine autophosphorylation of RIP2 as a readout of kinase activity. IVK assays were performed using RIP2 immunoprecipitated from FcγR-stimulated RAW 264.7 cells (F) and WT BMDMs (G) using an ADP-Glo assay. H, WT BMDMs were unstimulated or stimulated with murine anti-BSA IgG₁ + BSA for 4 h. RNA was extracted, and qRT-PCR was performed for previously defined genetic RIP2 activation markers. Data in graphs represent means ± S.D. Data are aggregated from at least three independent experiments using n = 3–7 mice for the unstimulated condition and n = 3–7 mice for the FcγR-stimulated condition. One-way ANOVA with Sidak's multiple comparisons test was used for statistical analysis of IVK assays and a Student's t test was used to analyze (log) -fold changes in gene expression (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001). RLU, relative luciferase units. Longer horizontal lines represent means. Error bars represent S.D.

Figure 2.

RIP2 is involved in signaling downstream of FcγR engagement. BMDMs from WT and RIP2-KO mice were stimulated with murine anti-BSA IgG₁ + BSA (A), murine anti-BSA IgG_2a + BSA (B), or murine IgG + anti-mIgG (C) for the indicated times. Western blotting was performed, and lysates were immunoblotted with the indicated antibodies. Data shown are representative of at least three independent experiments performed.

Figure 3.

RIP2 is not involved in FcγR-mediated cytokine production. BMDMs from WT and RIP2-KO mice were left unstimulated or were stimulated with murine anti-BSA IgG₁ + BSA for 4 h or for 16 h in low-serum medium. A, supernatants were collected after 16 h of stimulation for analysis of cytokine secretion by ELISA. B, in a separate set of experiments, RNA was harvested from cells after 4 h to perform qRT-PCR for determining expression of the indicated genes. Bars within graphs indicate means ± S.D. Data are aggregated from at least three independent experiments using n = 10 mice per group. Two-way ANOVA was used for statistical analysis. For A and B, no interaction was observed (cytokine secretion and gene up-regulation were similar for WT and RIP2-KO mice upon treatment). Therefore, no further testing was performed. The p value for the overall effect of FcγR stimulation is indicated within the graph (**, p < 0.01; ****, p < 0.0001). Longer horizontal lines represent means. Error bars represent S.D.

Figure 4.

RIP2 is not involved in FcγR-mediated phagocytosis. BMDMs from WT and RIP2-KO mice were incubated with an equal number of PKH26 Red–labeled sRBCs that were previously unopsonized or opsonized with rabbit IgG against sRBCs (1:1600). BMDMs were allowed to phagocytose sRBCs for 30 min at 37 °C. Unphagocytosed sRBCs were osmotically lysed, and BMDMs were washed and analyzed by flow cytometry. A, red fluorescence from phagocytosed sRBCs appears as a second peak to the right in flow cytometry histograms. Quantitation for flow cytometric analysis of phagocytosis is presented as percentage of cells positive for red fluorescence and is shown below the histograms. B, phagocytosis of PKH26-labeled opsonized sRBCs by WT and RIP2-KO BMDMs was also analyzed by confocal microscopy. Images of BMDMs incubated with unopsonized or opsonized PKH26-labeled sRBCs were taken after allowing phagocytosis to occur for 30 min. Quantitation for confocal analysis of phagocytosis is presented as particles per 100 macrophages (a total of three 40× fields were counted) shown beside the images. Bars within graphs represent means ± S.D. Data are aggregated from two independent experiments using n = 7 mice per group for flow cytometric analysis of phagocytosis. Data are aggregated from two independent experiments using n = 6 mice per group for confocal analysis of phagocytosis. Statistical analysis was performed using a two-way ANOVA. For A and B, no interaction was observed (phagocytosis of the PKH26-labeled opsonized sRBCs was found to be similar for WT and RIP2-KO mice). Therefore, no further testing was performed. The p value for the overall effect of FcγR stimulation is indicated within the graph (****, p < 0.0001). Mφ, macrophages. Longer horizontal lines represent means. Error bars represent S.D.

Figure 5.

RIP2 does not affect FcγR-mediated iNOS expression and nitrate/nitrite production. A, BMDMs from WT and RIP2-KO mice were primed with 100 ng/ml IFN-γ overnight prior to FcγR stimulation for 24 h. Cell lysates were then harvested to perform Western blotting. FcγR-mediated production of iNOS by WT and RIP2-KO BMDMs was assessed by immunoblotting using an anti-iNOS antibody and anti-tubulin antibody as a loading control. Three sets of BMDMs are shown for each genotype. B, BMDMs from WT and RIP2-KO mice were left unstimulated or were stimulated with murine anti-BSA IgG₁ + BSA. RNA was harvested from cells after 4 h to perform qRT-PCR to determine expression of iNOS. C, IFN-γ–primed WT and RIP2-KO BMDMs were left unstimulated or were stimulated with murine anti-BSA IgG₁ + BSA for 24 h. Cell supernatants were collected for assessment of nitrate/nitrite using a Griess assay. Bars within graphs represent means ± S.D. For qRT-PCR analysis of iNOS expression, data are aggregated from two independent experiments using n = 10 mice per group. For nitrate/nitrite quantification, data are aggregated from two independent experiments using n = 8 mice per group. Statistical analysis was performed using a two-way ANOVA. For B and C, no interaction was observed (iNOS expression and nitrate/nitrite production as a result of FcγR stimulation were found to be similar for WT and RIP2-KO mice). Therefore, no further testing was performed. The p value for the overall effect of FcγR stimulation is indicated within the graph (****, p < 0.0001). Longer horizontal lines represent means. Error bars represent S.D.

Figure 6.

RIP2 is involved in ROS production downstream of FcγR engagement. A, BMDMs from WT and RIP2-KO mice were primed with 100 ng/ml IFN-γ overnight prior to FcγR stimulation for 30 min in the presence of 2 μm oxidative stress detection probe. The probe reacts with various ROS to produce a green fluorescent product indicated by increased fluorescence in the FL-1 channel. Treatment of FcγR-stimulated cells with an ROS inhibitor, NAC, results in a leftward shift and a decrease in the FcγR-induced fluorescence. B, quantitation for flow cytometric analysis of ROS production is presented as percentage of cells with green fluorescence (compared with unstained control cells) or MFI in the FL-1 channel. Bars within graphs represent means ± S.D. Data are aggregated from three experiments using n = 11 mice per group. Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparisons test (**, b, p < 0.01; a, p < 0.0001 when compared with the FcγR-stimulated group of the same genotype). Longer horizontal lines represent means. Error bars represent S.D.

Figure 7.

Fgr directly tyrosine-phosphorylates RIP2. HEK293 cells were transiently cotransfected with kinase-dead RIP2 and either WT, constitutively active, or kinase-dead Fgr (A) or Hck (B). RIP2 was immunoprecipitated, and tyrosine phosphorylation was assessed. HEK293 cells were singly transfected with either kinase-dead RIP2 or with either WT or kinase-dead Fgr (C) or Hck (D). RIP2, Fgr, and Hck were immunoprecipitated individually and combined together in an IVK assay as indicated. HEK293 cells were transiently transfected with WT RIP2 with or without Fgr (E) or Hck (F). RIP2 was immunoprecipitated, and an IVK assay (for tyrosine autophosphorylation) was performed. Data presented are representative of at least three independent experiments performed.

Figure 8.

NOD2 is required for the observed effects of RIP2 in FcγR-mediated ROS production. BMDMs from WT and NOD2-KO mice were left unstimulated or were stimulated with murine anti-BSA IgG₁ + BSA for 4 or 16 h in low-serum medium. A, supernatants were collected after 16 h of stimulation for analysis of cytokine secretion by ELISA. B, in a separate set of experiments, RNA was harvested from cells after 4 h to perform qRT-PCR for determining expression of the indicated genes. C, BMDMs from WT and NOD2-KO mice were primed with 100 ng/ml IFN-γ overnight prior to FcγR stimulation for 30 min in the presence of 2 μm oxidative stress detection probe with or without addition of a ROS inhibitor, NAC. Quantitation for flow cytometric analysis of ROS production is presented as percentage of cells with green fluorescence (compared with unstained control cells) or MFI in the FL-1 channel. Bars within graphs represent means ± S.D. Data are aggregated from three experiments using n = 8 mice per group. Statistical analysis was performed using a two-way ANOVA with Tukey's multiple comparisons test. For A and B, no interaction was observed (cytokine secretion and gene up-regulation were similar for WT and NOD2-KO mice upon treatment). Therefore, no further testing was performed. The p value for the overall effect of FcγR stimulation is indicated within the graph (**, p < 0.01; ****, p < 0.0001; a, p < 0.0001 when compared with the FcγR-stimulated group of the same genotype).