Identification of a number of susceptibility loci for CD in the past several years constitutes significant progress in efforts to understand the pathogenesis of this complex disease. However, functional studies are required to understand the underlying pathophysiologic mechanisms reflected by these many susceptibility loci. IFN-γ is a key cytokine in immune responses. Plasma and mucosal concentrations of IFN-γ are increased in CD (23
). Anti–IFN-γ Abs have shown clinical benefit in CD in two independent studies (31
). Similarly, anti–IFN-γ showed protective effects in mice with anti-CD40–induced colitis (33
). These observations implicate the IFN-γ pathway in the pathogenesis of CD. In this report, we investigated the involvement in the IFN-γ response of LRRK2, whose locus is located downstream of an SNP associated with a higher risk of developing CD. We detected LRRK2 expression in immune cells and tissues, we identified LRRK2 as an IFN-γ target gene upregulated by bacteria-induced or CD inflammation, and we showed that LRRK2 is an activator of the NF-κB pathway. These observations suggest that LRRK2 might be involved in the regulation of mucosal IFN-γ immune responses that are relevant to host responses to pathogens as well as CD.
In nonstimulated conditions, we observed that human monocytes, macrophages, and B lymphocytes express the highest amount of LRRK2 mRNA, although, basal LRRK2 expression is low overall, which is consistent with previous reports (21
). However, LRRK2 is highly expressed after IFN-γ stimulation. This striking difference in expression between basal condition and upon IFN-γ stimulation suggests that LRRK2 transcription is tightly regulated in physiologic conditions. Previous reports have already shown LRRK2 expression in extrabrain tissues and in immune-related tissues, such as the spleen, thymus, and lymph nodes (21
). A recent study using mice showed that LRRK2 expression is the highest in the spleen and is detected in B cells and macrophages, but not in T cells, which is consistent with our data collected from intestinal human tissues (34
). In the context of IBD, the expression in lymphoid organs is especially interesting, because they contain immune cells involved in CD pathogenesis that are responsive to IFN-γ. The fact that LRRK2 is expressed in monocytes, macrophages, dendritic cells, and B cells and can be strongly upregulated by IFN-γ suggests a role for LRRK2 in immune functions. Many of the IFN-γ–induced functions implicated in pathogen resistance require activation of immune-signaling pathways. Our observations show that LRRK2 is able to activate inflammatory signaling pathways, suggesting that LRRK2 is likely involved in the transduction of the IFN-γ signal to downstream effectors. LRRK2 contains two catalytic domains that are functional in vitro and many protein interaction domains whose functions still need to be explored (9
). Therefore, LRRK2 is thought to be involved in multiprotein-signaling protein complexes. Because LRRK2 is a kinase, we investigated whether the activation of those immune signaling pathways requires the phosphorylation of LRRK2 substrates. Surprisingly, a dead-kinase mutant still induces NF-κB activation, indicating that activation of this pathway is independent of LRRK2 kinase activity. In LRRK2 knockdown experiments, we did not detect any consistent differences in IL-6, MCP-1, and COX-2 transcription activation following Salmonella
infection. As the intracellular PAMP receptor Nods, LRRK2 contains a leucin rich repeat domain, can form homodimer, and activates NF-κB in an overexpression setting that favors the homodimerization. We speculate that LRRK2 might have a specific stimulatory ligand (microbial or not) that is not expressed by S. typhimurium
and remains to be defined. This ligand will activate LRRK2 dimerization and enhance the effect of LRRK2 on NF-κB signaling.
Mice deficient in IFN-γ or IFN-γ receptor are hypersensitive to bacterial, parasitic, and viral infections (35
). A GWAS recently demonstrated the association of an SNP located upstream from the LRRK2 locus with a higher susceptibility to Mycobacterium leprae
). That study also highlighted other genes already known to be associated with CD, such as NOD2, TNFS15, and C13ORF31, suggesting that the pathways involved in the control of M. leprae
might be also involved in the susceptibility to CD. Interestingly, previous reports have suggested that M. avium
tuberculosis might be involved in CD (39
). Our data show that LRRK2 is recruited close to the pathogens during bacterial infection, and LRRK2 participates in the ROS production during the phagocytic burst and the antibacterial activity. These observations suggest that LRRK2 is likely to be involved in the host response to pathogens, particularly in the context of the IFN-mediated immune responses.
Because LRRK2 mutations are the most common genetic alteration associated with PD, it is reasonable to wonder about the potential relevance of the present findings to PD (6
). Although our results suggest that LRRK2 polymorphisms might be involved in immune responses, dysregulated in CD or in the host reponse to pathogen, the relevance of our findings regarding PD pathophysiology is uncertain. LRRK2-deficient mice exhibit no obvious phenotype (40
), but our findings support the potential relevance of assessing phenotype characterizations in the context of inflammation. Detection of LRRK2 at the protein level has been a challenge that limited the progress in the investigation of LRRK2 functions. For example, although a GWAS identified multiple SNPs associated with a higher risk of developing PD (41
), the authors reported their inability to perform quantitative trait analysis to detect differences in LRRK2 expression associated with the disease SNPs because of the insufficient expression at the probe for LRRK2. In this case, our findings indicate the potential relevance of investigating the association of PD-SNPs with LRRK2 expression in the context of IFN stimulation.
Of interest, and in addition to the intestinal macrophages reported in this study, LRRK2 protein has been detected in activated macrophage–microglia in the brain of patients with PD (7
). Although the main PD-associated mutant LRRK2-G2019S did not activate NF-κB differently from wild type protein, our data suggest that studies of PD should consider the potential relevance of inflammatory responses mediated by LRRK2 in the pathogenesis of PD. Several studies have suggested that inflammation might be involved in the pathophysiology of PD (42
), whereas our report provides evidence that LRRK2 has a role in immune responses mediated by IFN-γ.