Effect of LRRK2 protein and activity on stimulated cytokines in human monocytes and macrophages

Abstract

Leucine-rich-repeat kinase 2 (LRRK2), a potential therapeutic target for the treatment of Parkinson's disease (PD), is highly expressed in monocytes and macrophages and may play a role in the regulation of inflammatory pathways. To determine how LRRK2 protein levels and/or its activity modulate inflammatory cytokine/chemokine levels in human immune cells, isogenic human induced pluripotent stem cells (iPSC) with the LRRK2-activating G2019S mutation, wild-type LRRK2, and iPSC deficient in LRRK2 were differentiated to monocytes and macrophages and stimulated with inflammatory toll-like receptor (TLR) agonists in the presence and absence of LRRK2 kinase inhibitors. The effect of LRRK2 inhibitors and the effect of increasing LRRK2 levels with interferon gamma on TLR-stimulated cytokines were also assessed in primary peripheral blood-derived monocytes. Monocytes and macrophages with the LRRK2 G2019S mutation had significantly higher levels of cytokines and chemokines in tissue culture media following stimulation with TLR agonists compared to isogenic controls. Knockout of LRRK2 impaired phagocytosis but did not significantly affect TLR-mediated cytokine levels. Interferon gamma significantly increased the levels of LRRK2 and phosphorylation of its downstream Rab10 substrate, and potentiated TLR-mediated cytokine levels. LRRK2 kinase inhibitors did not have a major effect on TLR-stimulated cytokine levels. Results suggest that the LRRK2 G2019S mutation may potentiate inflammation following activation of TLRs. However, this was not dependent on LRRK2 kinase activity. Indeed, LRRK2 kinase inhibitors had little effect on TLR-mediated inflammation under the conditions employed in this study.

Fig. 1. Characterisation of IPS-derived monocytes.

Flow cytometry was used to measure the number of cells expressing the monocyte marker CD14 in collected tissue media following monocyte differentiation with representative flow cytometry plots shown in (a). Up to 100,000 events were generally captured. Flow cytometry results were then quantified (b), with the graph showing the mean percent of CD14 cells in the tissue culture media, while the joined dots indicate specific results for each genotype for the different differentiation rounds. Flow cytometry was also used to measure the expression of monocyte markers CD68 (c), HLA-DR (d), CCR2 (e) and CD163 (f) after gating on CD14-positive monocytes. Graphs show mean ± SEM with dots indicating triplicate measurements. Results are representative of at least 2 biological replicates. g CD14 and CD16 were used to determine the percent of classical, intermediate and non-classical monocytes in the different LRRK2 genotype cell lines. The uptake of GFP-labelled latex beads, shown in green in the representative micrographs was determined in differentiated macrophages using immunofluorescence at 3 h (h), and 16 h (i). Cell mask, shown in magenta, was used to visualise internalised GFP signal. The blue staining is DAPI. The scale bar = 20 μm. Graphs indicate the median fluorescence intensity (MFI) of GFP puncta per cell ±SEM. Dots indicate the results from each image analysed, with each image comprising ~20 cells. *Indicates P < 0.05. Protein lysates were generated from differentiated macrophages and subjected to immunoblot with representative images shown in (j) for the indicated proteins. Immunoblots were quantified for LRRK2 normalised to β-actin (k), T73 phosphorylated Rab10 normalised to total Rab10 (l) and S1292 phosphorylated LRRK2 normalised to total LRRK2 (m). Graphs show mean ± SEM with dots indicating biological replicates (n = 4). *Indicates P < 0.05.

Fig. 2. Potentiated cytokines in G2019S monocytes and macrophages.

Multiplex ELISA assay was used to measure levels of the indicated inflammatory cytokines in tissue culture media from LRRK2 wild type (WT, green bars), G2019S (blue bars) and knockout (KO, orange bars) monocytes following 24 h stimulation with 500 ng/ml LPS (a), 1 μg/ml pam3CSK4 (b) and 1 μg/ml CLO97 (c). d LRRK2 wild-type and G2019S monocytes were further differentiated to macrophages and cytokines in the tissue culture media were measured following 24 h stimulation with 500 ng/ml LPS. For all graphs, the data are expressed as the percent change for a particular cytokine relative to LRRK2 wild type, which was set at 100%. Graphs show mean ± SEM and are based on n = 3 technical replicates and representative of at least two biological replicates. *Indicates P < 0.05 for G2019S compared to wild type. ^#Indicates P < 0.05 for LRRK2 knockout compared to wild type.

Fig. 3. LRRK2 kinase inhibitors do not affect TLR-mediated inflammation.

a Multiplex ELISA assay was used to measure levels of the indicated inflammatory cytokines in tissue culture media from LRRK2 G2019S cells that were treated with DMSO (green bars), or the LRRK2 kinase inhibitors PF06447475 (0.5 μM, blue bars) or MLi2 (0.1 μM, orange bars) for 24 h following stimulation with 500 ng/ml LPS. b Immunoblotting of the LRRK2 pharmacodynamic biomarker site, serine 935 was performed in parallel sets of differentiated monocytes. c Multiplex ELISA assay was used to measure levels of the indicated inflammatory cytokines in tissue culture media from primary monocytes isolated from healthy donor peripheral blood mononuclear cells were cells that were treated with DMSO (green bars), or the LRRK2 kinase inhibitors PF06447475 (0.5 μM, blue bars) or MLi2 (0.1 μM, orange bars) for 24 h following stimulation with 500 ng/ml LPS. Immunoblotting was used to measure LRRK2 serine 935 (d) and Rab10 threonine 73 (e) phosphorylation in primary monocytes isolated from healthy blood donors and treated as above (n = 6). Representative immunoblots are shown. Graphs show mean ± SEM and data is expressed as the percent change relative to DMSO treated cells which are set at 100%. Cytokine graphs are based on n = 3 technical replicates and representative of at least two biological replicates. *P < 0.05 compared to DMSO.

Fig. 4. Interferon gamma increases LRRK2 levels and Rab10 phosphorylation.

Primary monocytes from healthy donors were differentiated to macrophages and then treated with 100 U/ml interferon gamma for 48 h. Immunoblot analysis was then performed for LRRK2 (a) and total and T73 phosphorylated Rab10 (b). Representative immunoblots are shown. Graphs show mean ± SEM (N = 6) and data is expressed as the percent change relative to untreated cells, which are set at 100%. **P < 0.01, ***P < 0.001 compared to untreated cells. c Primary monocytes were differentiated to macrophages and then treated with or without 100 U/ml interferon gamma for 24 h followed by treatment with or without 500 ng/ml LPS for a further 24 h. Multiplex ELISA assays were used to measure levels of the indicated inflammatory cytokines in tissue culture media. Graphs show mean ± SEM (n = 6). *P < 0.05, ***P < 0.001, compared to cells treated only with LPS. d Primary monocytes were differentiated to macrophages and then treated with 100 U/ml interferon gamma for 24 h followed by 500 ng/ml LPS for a further 24 h in the presence or absence of 0.1 μM MLi2. Multiplex ELISA assays were used to measure levels of the indicated inflammatory cytokines in tissue culture media. Graphs show data from the different individuals (n = 6). *P < 0.05 compared to DMSO. e Representative immunoblot of total and S935 phosphorylated LRRK2 following treatment with or without 0.1 μM MLi2.