Article LRRK2 promotes the activation of NLRC4 inflammasome during Salmonella Typhimurium infection Weiwei Liu,1* Xia’nan Liu,1* Yu Li,1 Junjie Zhao,3 Zhenshan Liu,1 Zhuqin Hu,1 Ying Wang,1 Yufeng Yao,2 Aaron W. Miller,3,4 Bing Su,1 Mark R. Cookson,5 Xiaoxia Li,3,6 and Zizhen Kang1,3,6 1Shanghai Institute of Immunology and 2Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai, China 3Department of Immunology and 4Department of Urology, Cleveland Clinic, Cleveland, OH 5Cell Biology and Gene Expression Section, Laboratory of Neurogenetics, National Institute on Aging, Bethesda, MD 6Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine, Case Western Reserve University, Cleveland, OH Although genetic polymorphisms in the LRRK2 gene are associated with a variety of diseases, the physiological function of LRRK2 remains poorly understood. In this study, we report a crucial role for LRRK2 in the activation of the NLRC4 inflam- masome during host defense against Salmonella enteric serovar Typhimurium infection. LRRK2 deficiency reduced caspase-1 −/− activation and IL-1β secretion in response to NLRC4 inflammasome activators in macrophages.Lrrk2 mice exhibited im- Downloaded from paired clearance of pathogens after acute S. Typhimurium infection. Mechanistically, LRRK2 formed a complex with NLRC4 in the macrophages, and the formation of the LRRK2–NLRC4 complex led to the phosphorylation of NLRC4 at Ser533. Impor- tantly, the kinase activity of LRRK2 is required for optimal NLRC4 inflammasome activation. Collectively, our study reveals an important role for LRRK2 in the host defense by promoting NLRC4 inflammasome activation. jem.rupress.org INTRODUCTION The leucine-rich repeat kinase 2 (LRRK2) gene is emerg- single nucleotide polymorphism, which results in an unstable ing as a genetic hotspot for disease associations. Pathogenic LRRK2 protein, has been shown to confer increased suscep- mutations in LRRK2 are the most prevalent genetic alter- tibility to leprosy, a disease caused by Mycobacterium leprae ations among Parkinson’s disease (PD) patients (Paisán-Ruíz infection (Zhang et al., 2009). In the mouse model, LRRK2 et al., 2004; Zimprich et al., 2004; Cookson, 2010), and single was required for the mucosal immunity against the Listeria on December 5, 2017 nucleotide polymorphisms in the LRRK2 gene have been monocytogenes (Zhang et al., 2015b). At the cellular level, linked to a variety of inflammatory diseases including Crohn’s LRRK2 was found to colocalize with intracellular Salmo- disease, ulcerative colitis, and cancer (Barrett et al., 2008; nella enteric serovar Typhimurium (S. Typhimurium) during Franke et al., 2010; Saunders-Pullman et al., 2010; Anderson bacterial infection in macrophages (Gardet et al., 2010). et al., 2011; Inzelberg et al., 2012). These epidemiological ev- These evidences collectively indicate that LRRK2 is directly idences have instigated intense research efforts focusing on involved in the innate immune response against intracellu- the pathogenic mechanisms of LRRK2 variants with the ul- lar bacteria. However, the molecular mechanism by which The Journal of Experimental Medicine timate goal of targeting LRRK2 for treatment. LRRK2 contributes to the host immunity is unknown. Despite the growing literature on the roles of the A major host response against the infection by intra- LRRK2 in disease development, much of its physiological cellular bacteria is the activation of NLRC4 inflammasome function remains elusive (Chia et al., 2014; Cookson, 2015). (Amer et al., 2006; Sutterwala et al., 2007; Suzuki et al., 2007; The expression pattern of LRRK2 points to a critical func- Case et al., 2009; Miao et al., 2010a). For example, S. Ty - tion in the immune system. LRRK2 can be induced by IFN-γ phimurium infection of macrophages induces NLRC4 in- stimulation in human monocytes, and it is preferentially ex- flammasome–mediated production of the proinflammatory pressed in mature macrophages and dendritic cells (Gardet et cytokines IL-1β and IL-18 (Franchi et al., 2006; Miao et al., al., 2010). Consistently, accumulating evidence suggests that 2010b). Activation of NLRC4 inflammasome is initiated LRRK2 plays an important role in the host defense against by the host recognition of cytosolic bacterial components the intracellular pathogens. In humans, an LRRK2 missense such as flagellin or PrgJ, triggering the oligomerization of NLRC4 proteins (Miao et al., 2010b; Zhao et al., 2011). The *W. Liu and X. Liu contributed equally to this paper. NLRC4 oligomers nucleate the filament formation of the Correspondence to Xiaoxia Li: [email protected]; Zizhen Kang: [email protected] adapter protein ASC (apoptotic speck protein containing a caspase recruitment domain) and protease caspase-1 (Hu et Abbreviations used: ASC, apoptotic speck protein containing a caspase recruitment domain; CARD, caspase activation and recruitment domain; DSS, disuccinimidyl su- berate; iBMDM, immortalized BMDM; LRR, leucine-rich repeat; MOI, multiplicity of © 2017 Liu et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike– No Mirror Sites license for the first six months after the publication date (see http://www .rupress .org / infection; PCF, peritoneal cavity–flushed fluid; PD, Parkinson’s disease; ROC, Ras of terms /). After six months it is available under a Creative Commons License (Attribution–Noncommercial– complex. Share Alike 4.0 International license, as described at https ://creativecommons .org /licenses /by -nc -sa /4 .0 /). Supplemental material can be found at: The Rockefeller University Press http://doi.org/10.1084/jem.20170014 J. Exp. Med. 2017 Vol. 214 No. 10 3051–3066 3051 https://doi.org/10.1084/jem.20170014 al., 2015; Zhang et al., 2015a). Oligomerization of caspase-1 LRRK2 is required for host defense against leads to proximity-induced proteolytic activation and subse- S. Typhimurium infection quently results in the maturation IL-1β and IL-18 (Vance, We then sought to determine whether LRRK2 deficiency 2015). Secreted IL-1β and IL-18 then recruit both the innate also reduces NLRC4 inflammasome activation in vivo. A re- and adaptive immune system for the clearance of pathogens cent study reported that NLRC4-dependent IL-1β produc- (Schroder and Tschopp, 2010). tion from the neutrophils was crucial for protecting mice from In this study, we report that LRRK2 is essential for the acute Salmonella infection delivered through intraperitoneal optimal activation of NLRC4 inflammasome duringS. Ty - injection (Chen et al., 2014). After a similar experimental phimurium infection. We found that the LRRK2-deficient design, we injected Lrrk2−/− and WT mice with 107 CFUs macrophages showed diminished caspase-1 activation and of S. Typhimurium intraperitoneally to induce peritonitis. reduced mature IL-1β secretion in response to NLRC4 in- IL-1β levels in the peritoneal cavity–flushed fluids (PCFs) flammasome activators. In addition,Lrrk2 −/− mice exhibited and in the sera were significantly reduced in theLrrk2 −/− impaired ability to clear the pathogens during acute S. Typh- mice compared with that in the littermate control (WT) imurium infection. Mechanistically, LRRK2 formed a com- mice 6 h after infection (Fig. 2 a). The reduction in IL-1β plex with NLRC4 in response to S. Typhimurium infection. production was associated with reduced infiltration of neu- Structure–function analysis showed that LRRK2 interacted trophils (Fig. 2 b). Furthermore, the neutrophils sorted from −/− with NLRC4 via the WD40 domain and that the kinase ac- infected Lrrk2 mice produced a smaller amount of IL-1β Downloaded from tivity of LRRK2 was required for full-scale caspase-1 activa- compared with WT controls (Fig. 2 c), suggesting impaired / tion and IL-1β secretion. Moreover, LRRK2 promoted the inflammasome activation inLrrk2 − − neutrophils. More- phosphorylation of NLRC4 at Ser533, a critical modification over, the reduction in IL-1β production was also associated required for the assembly of NLRC4 inflammasome. In sum- with increased bacteria colonization in the peritoneal cavity mary, our study discovered a novel role for LRRK2 in host (Fig. 2 d). A prior study showed that IL-1β is essential for defense against S. Typhimurium via promoting the activation controlling the bacteria infection in the S. Typhimurium–in- of the NLRC4 inflammasome. duced peritonitis model (Chen et al., 2014). Consistently, the jem.rupress.org bacterial colonization in the Lrrk2−/− mice was effectively at- RESULTS tenuated by supplementing the mice with recombinant IL-1β LRRK2 deficiency impairs NLRC4-dependent through intraperitoneal injection (Fig. 2 e). Furthermore, al- inflammasome activation though more than half of the WT mice remained alive 6 d To determine the role of LRRK2 in NLRC4 inflam- after bacteria inoculation, 90% of the Lrrk−/− mice died 4 d on December 5, 2017 masome activation, we first examined the caspase-1 activa- after infection (Fig. 2 f). Collectively, these data indicate that tion and IL-1β production in response to defined NLRC4 LRRK2 is required for the effective clearance of S. Typh- inflammasome activators inLRRK2 -deficient and WT imurium, which might be attributed to its role in promoting macrophages (Fig. 1, a–c and f). Cytosolic delivery of pu- NLRC4-mediated IL-1β production. rified flagellin or PrgJ, two NLRC4 inflammasome–specific ligands, induced robust pro–caspase-1 and pro–IL-1β cleav- LRRK2 deficiency reduces ASC speck formation upon