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. 2024 Nov 1;25(21):11754.
doi: 10.3390/ijms252111754.

RIPK2 Is Crucial for the Microglial Inflammatory Response to Bacterial Muramyl Dipeptide but Not to Lipopolysaccharide

Changjun Yang  1 , Maria Carolina Machado da Silva  1   2 , John Aaron Howell  1 , Jonathan Larochelle  1 , Lei Liu  1 , Rachel E Gunraj  1 , Antônio Carlos Pinheiro de Oliveira  1   2 , Eduardo Candelario-Jalil  1
Affiliations

RIPK2 Is Crucial for the Microglial Inflammatory Response to Bacterial Muramyl Dipeptide but Not to Lipopolysaccharide

Changjun Yang et al. Int J Mol Sci. .

Abstract

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is a kinase that is essential in modulating innate and adaptive immune responses. As a downstream signaling molecule for nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs), it is implicated in the signaling triggered by recognition of microbe-associated molecular patterns by NOD1/2 and TLRs. Upon activation of these innate immune receptors, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). However, whether RIPK2 is essential for downstream inflammatory signaling following the activation of NOD1/2, TLRs, or both remains controversial. In this study, we examined the role of RIPK2 in NOD2- and TLR4-dependent signaling cascades following stimulation of microglial cells with bacterial muramyl dipeptide (MDP), a NOD2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist. We utilized a highly specific proteolysis targeting chimera (PROTAC) molecule, GSK3728857A, and found dramatic degradation of RIPK2 in a concentration- and time-dependent manner. Importantly, the PROTAC completely abolished MDP-induced increases in iNOS and COX-2 protein levels and pro-inflammatory gene transcription of Nos2, Ptgs2, Il-1β, Tnfα, Il6, Ccl2, and Mmp9. However, increases in iNOS and COX-2 proteins and pro-inflammatory gene transcription induced by the TLR4 agonist, LPS, were only slightly attenuated with the GSK3728857A pretreatment. Further findings revealed that the RIPK2 PROTAC completely blocked the phosphorylation and activation of p65 NF-κB and p38 MAPK induced by MDP, but it had no effects on the phosphorylation of these two mediators triggered by LPS. Collectively, our findings strongly suggest that RIPK2 plays an essential role in the inflammatory responses of microglia to bacterial MDP but not to LPS.

Keywords: Toll-like receptor; inflammatory response; lipopolysaccharide; microglia; mitogen-activated protein kinase; muramyl dipeptide; nuclear factor-kappa B; nucleotide-binding oligomerization domain-like receptor; receptor-interacting serine/threonine protein kinase 2.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dose- and time-dependent degradation of RIPK2 by its proteolysis-targeting chimera in SIM-A9 cells. Model of RIPK2 degradation mediated by its proteolysis-targeting chimera (PROTAC) molecule GSK3728857A (A) and the molecular structure of the RIPK2 PROTAC (B). Representative immunoblots and graphs showing degradation of RIPK2 by RIPK2 PROTAC in microglial cells. (C,D) Incubation with various concentrations of RIPK2 PROTAC (0–10 μM) for 4 h degrades RIPK2 in a dose-dependent manner. (E,F) Similarly, time-dependent degradation of RIPK2 by RIPK2 PROTAC (1 μM) was also observed. One-way ANOVA with Bonferroni post-test; * p < 0.05 and *** p < 0.001 compared with control conditions. Data are normalized to β-actin and represented as mean ± SEM from three to four independent experiments.
Figure 2
Figure 2
MDP dose-dependently induces pro-inflammatory gene expression in SIM-A9 microglial cells. Graphs show treatment with various concentrations (0 to 10,000 ng/mL) of muramyl dipeptide (MDP) for 24 h dose-dependently increases transcription of pro-inflammatory genes Nos2 (A), Il-1β (B), Tnfα (C), Il6 (D) and Mmp9 (E) in microglial cells. qRT-PCR data are normalized to reference genes Cyc1 and Rltr2aiap and represented as fold increases compared to control cells. One-way ANOVA with Bonferroni post-test; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control conditions. Data are represented as mean ± SEM from four independent experiments.
Figure 3
Figure 3
RIPK2 PROTAC reduces MDP-induced pro-inflammatory gene expression and iNOS protein levels in SIM-A9 cells. SIM-A9 cells were pretreated with 1 µM RIPK2 PROTAC for 4 h followed by 20 h incubation with 100 ng/mL MDP. After that, cells were harvested for RNA and protein extraction. (A) Graph shows RIPK2 degradation by RIPK2 PROTAC completely reduced MDP-induced transcription of pro-inflammatory genes Nos2, Ptgs2, Il-1β, Tnfα, Il6, Ccl2, and Mmp9. (B–E) Effects of RIPK2 PROTAC on MDP-induced iNOS, COX-2, and RIPK2 protein levels. qRT-PCR data are normalized to reference genes Cyc1 and Rltr2aiap and represented as fold changes compared to MDP treatment, and immunoblot data are normalized to β-actin. One-way ANOVA with Bonferroni post-test; *** p < 0.001. Data are represented as mean ± SEM from three independent experiments.
Figure 4
Figure 4
RIPK2 PROTAC suppresses activation of both NF-κB p65 and MAPK p38 induced by MDP in SIM-A9 cells. (A) SIM-A9 cells were stimulated with 100 ng/mL MDP for indicated periods (0 to 120 min). Immunoblots show MDP increased phosphorylation of NF-κB p65 and MAPK p38, and 60 min incubation of MDP induced maximal effects on both phosphorylated protein levels. Data are representative of three independent experiments with similar results. (B–E) SIM-A9 cells were pretreated with 1 µM RIPK2 PROTAC for 4 h followed by 60 min incubation of 100 ng/mL MDP. After that, cells were harvested for protein extraction and Western blots. Immunoblots and graphs show that RIPK2 PROTAC completely abolished effects of MDP on phosphorylation of both NF-κB p65 (B,C) and MAPK p38 (B,D), which was associated with marked degradation of RIPK2 by its PROTAC pretreatment (B,E). One-way ANOVA with Bonferroni post-test; *** p < 0.001. Data are normalized to β-actin and represented as mean ± SEM from three independent experiments.
Figure 5
Figure 5
Effects of RIPK2 PROTAC on LPS-induced pro-inflammatory gene expression, protein levels of iNOS and COX-2, and phosphorylated NF-κB p65 and MAPK p38 levels in microglia. SIM-A9 microglial cells were pretreated with 1 µM RIPK2 PROTAC for 4 h followed by 20 h incubation with 10 ng/mL lipopolysaccharide (LPS). After that, cells were harvested for RNA and protein extraction. (A) Graph shows that RIPK2 PROTAC partly reduced LPS-induced Ptgs2, Il-1β, Il6, Ccl2, and Mmp9 gene transcription but did not affect gene expression of Nos2 and Tnfα. (B–E) RIPK2 PROTAC had no effects on LPS-induced increase in iNOS protein levels (B,C), but it slightly attenuated LPS-induced upregulation of COX-2 levels (B,D). Treatment with RIPK2 PROTAC had no effects on increased levels of phosphorylated NF-κB p65 (B,E) or p38 MAPK (B,F) induced by LPS. At same time, LPS-triggered upregulation of RIPK2 was potently degraded by its PROTAC (B,G). qRT-PCR data are normalized to reference genes Cyc1 and Rltr2aiap and represented as fold changes compared to LPS treatment, and immunoblot data are normalized to β-actin. One-way ANOVA with Bonferroni post-test; * p < 0.05, ** p < 0.01, and *** p < 0.001. Data are represented as mean ± SEM from three independent experiments.
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