Abstract
Background
Variants of the leucine-rich repeat kinase 2 (LRRK2) are associated with an increased susceptibility to Parkinson disease but also Crohn’s disease (CD). Studies using a murine colitis model have pointed to the involvement of LRRK2 in regulating an NFAT-dependent pathway that dampens the production of certain inflammatory cytokines. Also, recent work showed that LRRK2 interacts with NOD2 in Paneth cells in order to properly secrete antimicrobial peptides into the intestinal lumen and promote gut-microbiota homeostasis.
Aims
The present research is designed to develop a comprehensive understanding of the role of LRRK2 in immune system modulation, and how dysfunction of this pathway may lead to the development of Crohn’s disease (CD).
Methods
WT and LRRK2-deficient neutrophil and macrophages were infected with different Gram-negative and a Gram-positive Bacteria (S. typhimurium and Listeria monocytogenes) in a gentamicin protection assays and colony-forming unit assessment will determine the competence of LRRK2 deficient cells for bacterial phagocytosis (4h) and killing capacity (24h). We evaluate the ability of neutrophils from LRRK2-KO versus WT mice to transmigrate in vitro in a transwell assay using fMLP as a chemattractant. Also, we investigate the peritoneal cells (by FACS analysis) phenotype after injection of different microbial stimuli including FK105 (NOD1 ligand), MDP (NOD2 ligand) and LPS (TLR4 ligand) in WT mice compared to LRRK2-KO and G2019S-KI mice.
Results
We found that LRRK2 KO mice have a defect in migration of immune cells (neutrophil and monocytes) to the peritoneal cavity after injection of different microbial stimuli including FK105 (NOD1 ligand), MDP (NOD2 ligand) and LPS (TLR4 ligand). In contrast, the G2019S knock-in mice show a higher rate of migration of immune cells compared to cells from wild-type animals. Neutrophils from LRRK2 mice were compromised in their ability to transmigrate in vitro in a transwell assay using fMLP as a chemattractant. In parallel, we designed experiments to examine reactive oxygen species (ROS) produced in response to infection of myeloid cells with bacteria. Neutrophils and bone marrow-derived macrophages from LRRK2 KO mice infected with Listeria monocytogenes or Salmonella typhimurium were less able to restrict bacteria growth compared to WT cells. Consistent with these findings, cells from LRRK2 KO mice produced lower levels of ROS following bacterial infection. In order to determine whether myeloid cell migration is compromised in vivo during inflammation, we are beginning experiments in WT and KO mice looking at different models of ileitis/colitis.
Conclusions
With this work we will further characterize the role of LRRK2 in intestinal homeostasis and the mucosal barrier maintenance, including how its deficiency may predispose an individual to developing CD.
Funding Agencies
CAG, CIHR