LRRK2 mRNA expression in microarray datasets. Average microarray expression of the highest expressing LRRK2 probe across 79 human tissues obtained from the GNF consortium (left) and 34 tissues obtained from Riken RefDic (right). Expression values were sorted, and only the top 20 are shown. Bars are color coded to reflect the average value of Affymetrix present or absent calls. Black bars indicate that the transcript was identified as present.

LRRK2 is expressed in immune cells and is upregulated by IFN-γ. Quantitative RT-PCR was performed using cDNA from a Clontech (Mountain View, CA) human cDNA panel or generated from American Type Culture Collection (Manassas, VA) cell lines. Reaction products were loaded into acrylamide gels to check the presence of the unique amplicons corresponding to LRRK2 and GAPDH (left panel). Quantification of the relative amount of LRRK2 mRNA to GAPDH mRNA is shown on the right with normalization to the value obtained from THP-1 cells. THP-1 cells were differentiated into macrophages using PMA 400 nM for 24 h and then treated with TNF-α, IFN-γ (100 ng/ml), or peptidoglycan (10μg/ml). Total mRNA was extracted to quantify LRRK2 mRNA and GAPDH mRNA after 24 h using quantitative RT-PCR, or proteins were extracted to detect LRRK2 and actin by Western blot after 48 or 72 h. Before extractions, cells were observed by phase microscopy to ensure that stimulation did not induce cell death. Blood was collected from healthy donors, and PBMCs were isolated using Ficoll gradients. The following day, PBMCs were treated with IFN-γ (100 ng/ml) or left untreated for 48 h. mRNA was extracted to quantify LRRK2 mRNA and GAPDH mRNA using quantitative RT-PCR (left panel), or PBMCs were analyzed by flow cytometry to detect LRRK2 in CD3-, CD19-, and CD11b-positive cells (right panel). The displayed experiments are representative of three independent experiments. Statistical analyses were performed using Student t test (**p < 0.01).

LRRK2 expression in colonic biopsy specimens from patients with CD. A, Paired biopsy specimens from intestinal tissues of patients with IBD (inactive disease state) were collected: one specimen from an inflamed area and one from a control noninflamed area for each patient. Total mRNA was extracted and used for qRT-PCR. The amount of LRRK2 mRNA relative to GAPDH mRNA of each inflamed biopsy specimen was normalized to the quantity measured in the corresponding noninflamed biopsy specimen obtained from the same patient. Statistical analyses were performed using Student t test (*p < 0.05). B and C, Frozen sections (7 μm) were prepared from colonic biopsy specimens obtained from patients with IBD. Tissues were immunolabeled with rabbit anti-LRRK2 (clone 267) and rat anti–troma-1 Abs to detect intestinal epithelial cells. The image gallery in B was performed with anti-LRRK2 Abs preincubated or not with its corresponding blocking peptide. C, Mouse monoclonal anti-CD3, anti-CD19, anti-myeloperoxidase, anti-mannose receptor (MNR), and anti-CD103 Abs were also used to detect T-lymphocytes, B-lymphocytes, neutrophils, macrophages, and dendritic cells, respectively. After immunostaining using secondary FITC-anti–mouse, Cy3-anti–rabbit and Cy5-anti–rat species-specific Abs, fluorescence was detected by confocal microscopy. The image gallery displays single confocal sections (original magnification ×60). The bottom panel arrows indicate the cells containing signals from both LRRK2 and FITC-MNR, CD103, CD20, and CD3 cell specific markers, respectively.

NF-κB activation induced by LRRK2. HEK293T cells were transfected with reporter plasmids encoding firefly luciferase cloned under a promoter containing NF-κB elements (A, C, D) or AP-1 elements (B), and with a plasmid encoding Renilla luciferase as a transfection control at a ratio of 10:1. Transcriptional activation was quantified 24 h after transfection by ratios of firefly luciferase activity to Renilla luciferase activity. A, Luciferase plasmids were cotransfected with empty vector pcDNA3.1 and pcDNA3.1-LRRK2myc. NF-κB activation induced by LRRK2 was normalized to the activation induced by transfection of an empty vector with an equivalent amount. B, Luciferase plasmids for the AP-1 reporter assay were cotransfected with a positive control (k-rasV12), pcDNA-3.1-LRRK2myc, and their corresponding empty backbone vectors (0.5 μg). Activation was normalized to the activation induced by corresponding empty vectors. C, Luciferase plasmids were cotransfected with pc-DNA3.1-LRRK2myc (1 μg) and/or a dominant negative form of IKK-β (0.25 μg). Data were normalized to the activation induced by cotransfection of the corresponding empty backbone vectors. D, Luciferase plasmids were cotransfected with pcDNA3.1 plasmids empty or encoding for LRRK2-myc, kinase-dead mutant LRRK2 K1906M or PD-associated mutant LRRK2 G2019S (1 μg). Data were normalized to the activation induced by the empty vector. Statistical analyses were performed using Student t test (**p < 0.01).

LRRK2 participates in ROS production during phagocytosis and in antibacterial response. Raw 264.7 cells were transfected with siRNA control or two different siRNA-directed against-mouse LRRK2 sequence. After 48 h of transfection, cells were stimulated with IFN-γ or left unstimulated for 24 h. LRRK2 expression was determined by Western blot (A) and quantitative PCR (B) 48 and 72 h after transfection, respectively. Cells were incubated with luminol and with opsonised-zymosan to assess the production of ROS upon phagocytosis (C). Cells were subjected to bacterial infection using S. typhimurium for 40 min and incubated with gentamicin to kill extracellular bacteria. Surviving intracellular bacteria were recovered after a total of 2 h of infection and quantified as detailed in Materials and Methods. Statistical analyses were performed using Student t test (*p < 0.05).

LRRK2 colocalizes with ds-red expressing S. typhimurium during bacterial infection of macrophages. Raw 264.7 cells were treated with IFN-γ (100 ng/ml) for 24 h or left untreated and were then infected using S. typhimurium expressing dsRed for 30 min. Cells were washed using PBS and fixed using paraformaldehyde before LRRK2 immunofluorescent staining using rabbit anti-LRRK2 Ab (clone AL106) and and FITC anti-rabbit Ab. Fluorescence was detected by confocal microscopy (original magnification ×60). Image gallery displays single confocal sections.