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. 2018 Jun 6;10(444):eaan8162.
doi: 10.1126/scitranslmed.aan8162.

An increase in LRRK2 suppresses autophagy and enhances Dectin-1-induced immunity in a mouse model of colitis

Tetsuya Takagawa  1   2 , Atsushi Kitani  2 , Ivan Fuss  2 , Beth Levine  3 , Steven R Brant  4 , Inga Peter  5 , Masaki Tajima  2 , Shiro Nakamura  1 , Warren Strober  6
Affiliations

An increase in LRRK2 suppresses autophagy and enhances Dectin-1-induced immunity in a mouse model of colitis

Tetsuya Takagawa et al. Sci Transl Med. .

Abstract

The LRRK2/MUC19 gene region constitutes a high-risk genetic locus for the occurrence of both inflammatory bowel diseases (IBDs) and Parkinson's disease. We show that dendritic cells (DCs) from patients with Crohn's disease (CD) and lymphoblastoid cell lines derived from patients without CD but bearing a high-risk allele (rs11564258) at this locus as heterozygotes exhibited increased LRRK2 expression in vitro. To investigate the immunological consequences of this increased LRRK2 expression, we conducted studies in transgenic mice overexpressing Lrrk2 and showed that these mice exhibited more severe colitis induced by dextran sodium sulfate (DSS) than did littermate control animals. This increase in colitis severity was associated with lamina propria DCs that showed increased Dectin-1-induced NF-κB activation and proinflammatory cytokine secretion. Colitis severity was driven by LRRK2 activation of NF-κB pathway components including the TAK1 complex and TRAF6. Next, we found that membrane-associated LRRK2 (in association with TAB2) caused inactivation of Beclin-1 and inhibition of autophagy. HCT116 colon epithelial cells lacking Beclin-1 exhibited increased LRRK2 expression compared to wild-type cells, suggesting that inhibition of autophagy potentially could augment LRRK2 proinflammatory signaling. We then showed that LRRK2 inhibitors decreased Dectin-1-induced TNF-α production by mouse DCs and ameliorated DSS-induced colitis, both in control and Lrrk2 transgenic animals. Finally, we demonstrated that LRRK2 inhibitors blocked TNF-α production by cultured DCs from patients with CD. Our findings suggest that normalization of LRRK2 activation could be a therapeutic approach for treating IBD, regardless of whether a LRRK2 risk allele is involved.

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Conflict of interest statement

Competing interests: T.T., A.K., I.F., and W.S. are co-inventors on patent application no. US 20170079987 A1 “Treatment or prevention of an intestinal disease or disorder.” The other authors declare that they have no competing interests.

Figures

Fig. 1.
Fig. 1.. An increase in LRRK2 boosts inflammation in Lrrk2 Tg mice treated with DSS.
(A) LRRK2 mRNA expression in DCs from patients with CD. (B) LRRK2 mRNA expression in LCLs from individuals with genotype G/G or G/A at SNP rs11564258 [a nonrisk allele (G) and a risk allele (A)]. (C) LRRK2 protein relative to β-actin in CD patients with the same genotypes as in (B). (A and B) LRRK2 mRNA was measured by real-time polymerase chain reaction (PCR). (C) LRRK2 protein relative to β-actin was measured by Western blotting (fig. S1). (A) G/G genotype, n = 13; G/A genotype, n = 7, P = 0.0104; (B) G/G genotype, n = 9; G/A genotype, n = 9, P = 0.04; (C) G/G genotype, n = 9; G/A genotype, n = 9, P = 0.0098. (D) Lrrk2 Tg mice and littermate control mice were fed 2% DSS for 8 days, and body weight was measured. The data represent the average value for each group. n = 12 Lrrk2 Tg mice (Lrrk2 Tg) and n = 14 control mice (day 10, P = 0.0283; day 11, P = 0.0186). (E) Histological score of colon inflammation for Lrrk2 Tg and control mouse groups analyzed in (C). n = 12 Lrrk2 Tg mice and n = 14 control mice (P = 0.0054). Asterisk (*) indicates P < 0.05. (F) Representative photomicrographs of hematoxylin and eosin (H&E)–stained colon tissue on day 11 from Lrrk2 Tg mice and littermate controls. Data are presented as means ± SEM. Statistical significance was determined using an unpaired two-tailed Student’s t test.
Fig. 2.
Fig. 2.. LRRK2 positively regulates Dectin-1 signaling and interacts with the TAK1 complex.
(A and B) BMDCs from Lrrk2 Tg and control mice were stimulated for 24 hours with the indicated ligands in vitro. The amount of TNF-α in the culture supernatant was measured by enzyme-linked immunosorbent assay (ELISA). (A) Lrrk2 Tg mice, n = 3; control mice, n = 3, P = 0.0263; (B) Lrrk2 Tg mice, n = 3; control mice, n = 3; P = 0.0179 (ZymD), P = 0.0431 [heat-killed S. cerevisiae (HKSC)], and P = 0.0327 [heat-killed Candida albicans (HK-CA)]. (C) DCs from patients with CD (with the G/G or G/A genotype at SNP rs11564258) were stimulated for 24 hours with ZymD in vitro. The production of TNF-α in the culture super-natant was measured by ELISA (G/G genotype, n = 13; G/A genotype, n = 6, P = 0.026). (D) Whole-cell lysates (WCLs) of HEK293T cells cotransfected with the indicated plasmids were subjected to immunoprecipitation with anti- FLAG antibody followed by immunoblotting (IB) with anti– green fluorescent protein (GFP) antibody. IgG, immunoglobulin G. (E) Whole-cell lysates of HEK293T cells cotransfected with the indicated plasmids were subjected to immunoprecipitation (IP) with anti- GFP anti body, followed by immunoblotting with anti- hemagglutinin (HA) antibody. (F and G) Whole-cell lysates of HEK293T cells cotransfected with the indicated plasmids were subjected to immunoprecipitation with anti- FLAG antibody, followed by immunoblotting with anti- HA antibody. (H) Nuclear or cytoplasmic lysates of BMDCs from Lrrk2 Tg mice and littermate control mice were stimulated with ZymD in culture and then were subjected to immunoblotting with antibodies specific to the indicated ligands. Each of the studies is representative of at least three replicates. *P < 0.05 was considered a statistically significant difference. Statistical significance was determined with a Student’s t test. LPS, lipopolysaccharide; PGN, peptidoglycan; IKKα, IκB kinase α; MW, molecular weight.
Fig. 3.
Fig. 3.. LRRK2 interacts with Beclin-1 and suppresses autophagy.
(A and B) BMDCs from Lrrk2 Tg mice (Lrrk2 Tg) or littermate control mice were cultured with or without BafA1 (300 nM) for 30 min and then were stimulated with M. leprae for 2 hours. The cell lysates were subjected to immunoblotting to examine autophagic flux. Total RNA was extracted from BMDCs, and p62 mRNA expression was determined using real-time PCR. (B) Lrrk2 Tg mice, n = 3; control mice, n = 3; P values from left to right: P = 0.6819, P = 0.9586, P = 0.3713, and P = 0.3073). NS, not significant. (C) BMDCs from Lrrk2 Tg mice and littermate control mice that had been crossed with GFP-LC3 Tg mice were stimulated with M. leprae and then were evaluated for the number of GFP-LC3 puncta by confocal laser scanning microscopy. The data shown consist of pooled data from three independent experiments. (C) Lrrk2 Tg mice, n = 3; control mice, n = 3; M. leprae: 6 hours, P = 0.0010; M. leprae: 16 hours, P < 0.0001. *P < 0.05 was considered a statistically significant difference. Statistical significance was determined using a Student’s t test. (D) BMDCs from Lrrk2 Tg mice were stimulated with HK-CA for 60 min in culture and then were stained with anti-FLAG antibody (green) and anti-LAMP1 antibody (red). DIC, differential interference contrast. (E) Whole-cell lysates of HEK293T cells cotransfected with GFP-LRRK2 and FLAG–Beclin-1 plasmids were subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-GFP antibody. (F) BMDCs from Lrrk2 Tg mice cultured with or without HK-CA were subjected to a proximity ligation assay (PLA) to detect LRRK2/Beclin-1 complexes (red). PLA-, no PLA probe, negative control; DAPI, 4′,6-diamidino-2-phenylindole.
Fig. 4.
Fig. 4.. LRRK2 augments Beclin-1 degradation blocking autophagy and increasing LRRK2 expression.
(A) BMDCs from Lrrk2 Tg mice or littermate control mice were stimulated for 6 hours with M. leprae. The cell lysates were then subjected to immunoblotting with the anti–Beclin-1 antibody H-300. (B) Whole-cell lysates of HEK293T cells cotransfected with the indicated plasmids were sonicated and subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with anti-HA antibody. Immunoblotting using anti–Beclin-1 antibody (clone: 20/Beclin) demonstrated degradation bands (black arrows). (C and D) LRRK2 mRNA and protein expression was determined using real-time PCR (C) and immunoblotting (D) in HCT116 WT cells, Beclin-1 KO cells, and ATG5 KO cells. Data are representative of at least three identical experiments. (C) HCT116 WT versus Beclin-1 KO, P = 0.0009; HCT116 WT versus ATG5 KO, P = 0.0015. *P < 0.05 was considered a statistically significant difference. Statistical significance was determined by analysis of variance (ANOVA) for multiple comparisons.
Fig. 5.
Fig. 5.. LRRK2 inhibitors ameliorate gut inflammation in Lrrk2 Tg mice with DSS-induced colitis.
(A) BMDCs from Lrrk2 Tg mice or littermate control mice were cultured with dimethyl sulfoxide (DMSO) vehicle or the indicated LRRK2 inhibitor for 30 min in vitro and then stimulated with ZymD for 24 hours. Mouse TNF-α concentrations in culture supernatants were determined using ELISA. (B) DCs derived from PBMCs from eight CD patients were cultured with DMSO vehicle or the indicated LRRK2 inhibitor for 30 min in vitro and then stimulated with ZymD for 24 hours. Human TNF-α in culture supernatants was measured by ELISA. Inhibition of TNF-α was observed with four of seven LRRK2 inhibitors (P < 0.03, unpaired one-tailed t test). (C to E) Lrrk2 Tg mice or littermate control mice treated with DMSO vehicle or the LRRK2 inhibitor GNE-7915 (20 mg/kg) were administered 2% DSS for 8 days. (C)Changes in body weight as a percentage of untreated animals, (D) histological score, and (E) representative H&E staining of mouse colon epithelial tissue are shown. Control + DMSO, n = 19; control + GNE-7915, n = 8; Lrrk2 Tg + DMSO, n = 16; Lrrk2 Tg + GNE-7915, n = 16. All experiments were repeated at least three times. (C) Control + DMSO, n = 19; control + GNE-7915, n = 8; Lrrk2 Tg mice + DMSO, n = 16; Lrrk2 Tg mice +GNE-7915, n = 16; P values determined on day 10: control + DMSO versus control + GNE-7915, P = 0.0356; control + DMSO versus Lrrk2 Tg mice + DMSO, P = 0.0070; Lrrk2 Tg mice + DMSO versus Lrrk2 Tg mice + GNE-7915, P = 0.0208. (D) Control + DMSO versus control + GNE-7915, P = 0.0127; control + DMSO versus Lrrk2 Tg mice + DMSO, P = 0.0150; Lrrk2 Tg mice + DMSO versus Lrrk2 Tg mice + GNE-7915, P = 0.0001. Statistical significance was determined using ANOVA for multiple comparisons. Asterisk (*) indicates P < 0.05.
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