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. 2017 Jan;8(1):55-66.
doi: 10.1007/s13238-016-0326-x. Epub 2016 Nov 9.

LRRK2 Enhances Nod1/2-mediated Inflammatory Cytokine Production by Promoting Rip2 Phosphorylation

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Free PMC article

LRRK2 Enhances Nod1/2-mediated Inflammatory Cytokine Production by Promoting Rip2 Phosphorylation

Ruiqing Yan et al. Protein Cell. .
Free PMC article

Abstract

The innate immune system is critical for clearing infection, and is tightly regulated to avert excessive tissue damage. Nod1/2-Rip2 signaling, which is essential for initiating the innate immune response to bacterial infection and ER stress, is subject to many regulatory mechanisms. In this study, we found that LRRK2, encoded by a gene implicated in Crohn's disease, leprosy and familial Parkinson's disease, modulates the strength of Nod1/2-Rip2 signaling by enhancing Rip2 phosphorylation. LRRK2 deficiency markedly reduces cytokine production in macrophages upon Nod2 activation by muramyl dipeptide (MDP), Nod1 activation by D-gamma-Glu-meso-diaminopimelic acid (iE-DAP) or ER stress. Our biochemical study shows that the presence of LRRK2 is necessary for optimal phosphorylation of Rip2 upon Nod2 activation. Therefore, this study reveals that LRRK2 is a new positive regulator of Rip2 and promotes inflammatory cytokine induction through the Nod1/2-Rip2 pathway.

Keywords: Inflammation; LRRK2; NF-κB activation; Nod2; Rip2.

Figures

Figure 1
Figure 1
LRRK2 modulates cytokine production in BMDMs. (A–C) The relative fold changes of IL-6 (A), TNF-α (B), and IL-1β (C) mRNA transcripts in WT, Nod2 −/−, Rip2 −/− and Lrrk2 −/− BMDMs treated with 5 ng/mL LPS and 1 or 10 μg/mL MDP for 4 h, or mock-treated. (D–F) The amounts of IL-6 (D), TNF-α (E), and IL-1β (F) in supernatants from WT, Nod2 −/−, Rip2 −/− and Lrrk2 −/− BMDMs treated with 5 ng/mL LPS and 1 or 10 μg/mL MDP for 12 h, or mock-treated. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, NS denotes not significant, by Student’s t-test. Data are representative of three independent experiments (A–F)
Figure 2
Figure 2
LRRK2 modulates NF-κB activation in BMDMs. The levels of phosphorylated IκBα (A) and phosphorylated P65 (B) were monitored in WT and Lrrk2 −/− BMDMs treated with 5 ng/mL LPS and 1 or 10 μg/mL MDP for the indicated times. The total amounts of IκBα (A) and P65 (B) were also analyzed. Actin was used as a loading control. Data are representative of three independent experiments (A, B)
Figure 3
Figure 3
LRRK2 modulates MAPK pathways in BMDMs. The levels of phosphorylated ERK1/2 (A), phosphorylated P38 (B) and phosphorylated JNK (C) were determined by immunoblotting of lysates from WT and Lrrk2 −/− BMDMs treated with 5 ng/mL LPS and 1 or 10 μg/mL MDP for the indicated times. The total amounts of ERK1/2 (A), P38 (B) and JNK (C) were also analyzed. Actin was used as a loading control. Data are representative of three independent experiments (A–C)
Figure 4
Figure 4
LRRK2 modulates TAK1 activation in BMDMs. The amounts of phosphorylated TAK1 and total TAK1 were determined by immunoblotting of lysates from WT and Lrrk2 −/− BMDMs treated with 5 ng/mL LPS and 1 or 10 μg/mL MDP for the indicated times. Actin was used as a loading control. Data are representative of three independent experiments
Figure 5
Figure 5
LRRK2 affects phosphorylation of Rip2 in human macrophages. (A) Immunoblotting of phosphorylated murine Rip2 (S176) in WT, Nod2 −/−, and Rip2 −/− BMDMs using the commercial antibody against phosphorylated human (S176). BMDMs were treated with 5 ng/mL LPS and 10 μg/mL MDP for 30 minutes or mock-treated. Total Rip2 was immunoblotted as a control. Actin was used as a loading control. (B) Immunoblotting analysis of LRRK2 in control THP-1 cells or THP-1 cells with CRISPR/Cas9 knockout of LRRK2. (C) The amounts of phosphorylated Rip2 and total Rip2 were determined by immunoblotting of lysates from control THP-1 cells and THP-1 cells with LRRK2 knockout (KO #1) treated with 5 ng/mL LPS and 1 or 10 μg/mL MDP for the indicated times. Actin was used as a loading control. Data are representative of three independent experiments (A–C)
Figure 6
Figure 6
LRRK2 enhances the phosphorylation of Rip2 at Ser176. (A) The level of phosphorylated-Ser176 Rip2 was determined by immunoblotting of lysates from HEK293T cells transfected with WT LRRK2 or kinase-dead LRRK2 (K1906M) together with the Rip2 variants WT, K47M or S176A, or empty vectors. The levels of total Rip2 and LRRK2 were analyzed at the same time. (B) Relative NF-κB activity was analyzed using a dual luciferase assay in HEK293T cells transfected with WT LRRK2 or the kinase-dead LRRK2 mutant (K1906M) together with the Rip2 variants WT, K47M or S176A, or empty vectors. Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, NS denotes not significant, by Student’s t-test. (C–E) Co-immunoprecipitation analysis of the interaction between overexpressed LRRK2 and Rip2. Tagged LRRK2 and Rip2 were overexpressed in HEK293T cells, immunoprecipitated (IP) with anti-Myc (C) or anti-GFP (D) beads and immunoblotted with designated antibodies. (E) Co-immunoprecipitation analysis of the interaction between endogenously expressed LRRK2 and Rip2. Control THP-1 cells and THP-1 cells with LRRK2 knockout (ΔLRRK2 #1) were treated with 5 ng/mL LPS and 10 μg/mL MDP for 4 h, or mock-treated, immunoprecipitated with anti-LRRK2 antibody. Rip2 was immunoblotted in input fraction and immunoprecipitated fraction. Data are representative of three independent experiments (A–E)
Figure 7
Figure 7
LRRK2 modulates cytokine production during ER stress. (A–C) The relative fold changes of IL-6 (A), TNF-α (B), IL-1β (C) mRNA transcript levels in WT, Nod2 −/−, Rip2 −/− and Lrrk2 −/− BMDMs treated with 1 or 10 μmol/L Thapsigargin for 24 h, or mock-treated. (D) The amounts of IL-6 in supernatants from WT, Nod2 −/−, Rip2 −/− and Lrrk2 −/− BMDMs treated as in (A). Data are expressed as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, NS denotes not significant, by Student’s t-test. (E) The levels of phosphorylated Rip2 and total Rip2 were immunoblotted in control THP-1 cells and THP-1 cells with LRRK2 knockout (ΔLRRK2 #1) treated with 1 or 10 μmol/L Thapsigargin for 24 h. Actin was used as loading controls. Data are representative of three independent experiments (A-E)

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References

    1. Bertrand MJ, Doiron K, Labbe K, Korneluk RG, Barker PA, Saleh M. Cellular inhibitors of apoptosis cIAP1 and cIAP2 are required for innate immunity signaling by the pattern recognition receptors NOD1 and NOD2. Immunity. 2009;30:789–801. doi: 10.1016/j.immuni.2009.04.011. - DOI - PubMed
    1. Caruso R, Warner N, Inohara N, Nunez G. NOD1 and NOD2: signaling, host defense, and inflammatory disease. Immunity. 2014;41:898–908. doi: 10.1016/j.immuni.2014.12.010. - DOI - PMC - PubMed
    1. Damgaard RB, Nachbur U, Yabal M, et al. The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity. Mol Cell. 2012;46:746–758. doi: 10.1016/j.molcel.2012.04.014. - DOI - PubMed
    1. Dorsch M, Wang A, Cheng H, et al. Identification of a regulatory autophosphorylation site in the serine-threonine kinase RIP2. Cell Signal. 2006;18:2223–2229. doi: 10.1016/j.cellsig.2006.05.005. - DOI - PubMed
    1. Franke A, McGovern DP, Barrett JC, et al. Genome-wide meta-analysis increases to 71 the number of confirmed Crohn’s disease susceptibility loci. Nat Genet. 2010;42:1118–1125. doi: 10.1038/ng.717. - DOI - PMC - PubMed

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