The R1441C-Lrrk2 mutation induces myeloid immune cell exhaustion in an age- and sex-dependent manner in mice

Abstract

Age is the greatest risk factor for many neurodegenerative diseases, yet immune system aging, a contributor to neurodegeneration, is understudied. Genetic variation in the LRRK2 gene affects risk for both familial and sporadic Parkinson's disease (PD). The leucine-rich repeat kinase 2 (LRRK2) protein is implicated in peripheral immune cell signaling, but the effects of an aging immune system on LRRK2 function remain unclear. We analyzed peritoneal macrophages from R1441C-Lrrk2 knock-in mice and observed a biphasic, age-dependent effect of the R1441C-Lrrk2 mutation on peritoneal macrophage function. We report increases in antigen presentation, anti-inflammatory cytokine production, lysosomal activity, and pathogen uptake in peritoneal macrophages from young (2- to 3-month-old) female R1441C-Lrrk2 mice. Conversely, macrophages from aged (18- to 21-month-old) female R1441C-Lrrk2 mice exhibited decreased antigen presentation after inflammatory insult, decreased lysosomal function, and pathogen uptake, with a concomitant increase in DNA fragmentation in the presence of pathogens. This immune cell exhaustion phenotype was not observed in male R1441C-Lrrk2 mice and was driven by increased LRRK2 protein kinase activity. This phenotype was also observed in human peripheral myeloid cells, with monocyte-derived macrophages from patients with PD who had either the R1441C- or Y1699C-LRRK2 mutation exhibiting decreased pathogen uptake and increased PDL1 expression, consistent with immune cell exhaustion. Our findings that LRRK2 mutations conferred an immunological advantage at a young age but could predispose the carrier to age-acquired immune cell exhaustion have implications for the therapeutic development of LRRK2 inhibitors.

Figure 1.. R1441C-Lrrk2 mutation leads to age-dependent biphasic alteration in antigen presentation in pMacs.

pMacs from 2–3-month old and 18–21 month old female or male R1441C-Lrrk2 knockin mice or C57Bl/6 (B6) wildtype control mice were administered 100 units of IFNγ for 18 hours. (A, B) Total Lrrk2 protein expression was assessed and normalized to β-Actin and quantified. Representative western blots are shown. (C, D) Expression of Rab10 T73 protein (LRRK2 kinase substrate) was assessed and normalized to total Rab10 protein and quantified. Representative western blots are shown. (E, F) Surface MHC-II median fluorescence intensity (MFI) was quantified on LPMs by flow cytometry. (G-J) pMacs were stained for intracellular and extracellular MHC-II expression after treatment with 100 units of IFNγ for 18 hours in the presence or absence of the LRRK2 inhibitor PF360, and MFI and Ex:IcMHC-II ratio were quantified. This ratio measures the relative expression of intracellular and extracellular MHC-II. Scale bars, 100μm. Bars represent mean +/− SEM (n = 6–12 mice per group). Three-way ANOVA, Bonferroni post hoc. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, ****= p < 0.001.

Figure 2.. R1441C-Lrrk2 mutation leads to age-dependent biphasic alteration in cytokine release ex vivo and circulating cytokines in vivo.

(A, B) pMacs from 2–3-month old and 18–21-month old female or male R1441C-Lrrk2 or B6 control mice were stimulated with 100 units of IFNγ for 18 hours and culture medium was collected. Concentrations of IL10, IL6 and TNF in culture media were quantified and normalized to live cell count. (C, D) Blood from mice was collected via trunk bleed and plasma was isolated. Concentrations of IL10, IL6 and TNF were quantified. Bars represent mean +/− SEM (n = 8–10 mice per group).Three-way ANOVA, Bonferroni post hoc. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, ****= p < 0.001.

Figure 3.. R1441C-Lrrk2 mutation leads to age-dependent biphasic alterations in lysosomal activity in pMacs.

(A, B) pMacs from 2–3-month old and 18–21-month old female or male R1441C-Lrrk2 mice or B6 control mice were stimulated with 100 units of IFNγ for 18 hours. Cells were co-treated with 100nM PF360 vehicle and median fluorescence intensity (MFI) in the YAe assay was quantified for LPMs by flow cytometry. (C, D) MFI staining for DQ-BSA, a marker for lysosomal protein degradation, and BMV109, a pan-cathepsin fluorescent probe, was quantified in LPMs after treatment with 100 units of IFNγ for 18 hours by flow cytometry. (E-H) pMacs were treated with vehicle or 100nM PF30 for 2 hours and DQ-BSA and BMV109 MFI was quantified by microscopy. Scale bars, 100μM. Bars represent mean +/− SEM (n = 6–12 mice per group). Two -way ANOVA, Bonferroni post hoc, or Student’s t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, ****= p < 0.001.

Figure 4.. R1441C-Lrrk2 mutation alters pathogen uptake in pMacs in an age- and sex-dependent manner.

(A, B) pMacs from 2–3 month old and 18–21 month old female R1441C-Lrrk2 mice or B6 control mice were incubated with 100μg of pHrodo (a pH-sensitive fluorescent protein) E. coli with or without 100nM PF360. pMacs were imaged every 20 minutes for 5 hours, and green fluorescent protein (GFP) fluorescence was measured. Scale bars, 100μm. Four-way ANOVA, Bonferroni post hoc. Main effects of groups are reported in the graph key. (C) TUNEL assay MFI was quantified in LPMs by flow cytometry. Bars represent mean +/− SEM (n = 4–12 mice per group). One-way ANOVA, Bonferroni post hoc. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, ****= p < 0.001.

Figure 5.. R1441C-Lrrk2 mutation induces immune cell exhaustion in a kinase-dependent manner.

(A, B) pMacs from 2–3 month old and 18–21 month old female R1441C-Lrrk2 mice or B6 control mice were incubated with 100ng/mL of LPS for 5 days to induce immune cell exhaustion, with or without 100nM PF360. Lrrk2 protein expression was assessed and normalized to β-Actin and quantified. Representative western blots are shown. (C) PD-L1⁺ LPM counts were quantified by flow cytometry. (D) After 5 days of treatment with LPS or vehicle, pMacs were stimulated with 100ng/mL of IFNγ for 18 hours and MHC-II expression (MFI) was quantified by flow cytometry. Bars represent mean +/− SEM (n = 6–12 mice per group). Three-way ANOVA, Bonferroni post hoc. (E-G) After 5 days of LPS or vehicle treatment, pMacs were incubated with 100μg of pHrodo E. coli with or without 100nM PF360. pMacs were imaged every 20 minutes for 5 hours, and GFP⁺ cells (% of total) and GFP MFI were measured. Scale bars, 100μm. Four-way ANOVA, Bonferroni post hoc. Main effects of groups are reported in each graph key. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, ****= p < 0.001.

Figure 6.. R1441C-Lrrk2 mutation alters mitochondrial and oxidative stress gene transcription in macrophages from aged female mice.

(A-C) Transcriptomic analysis was conducted on IFNγ-treated pMacs isolated from 2–3-month old or18–21-month-old female and male R1441C-Lrrk2 mice or B6 control mice. Differentially expressed genes (DEGs) in IFNγ-treated pMacs from old versus young female mice were counted and compared between R1441C-Lrrk2 and B6 control groups. Volcano plots show genes with log₂-transformed fold change > 0.5 and an adjusted p-value ≤ 0.05. (D) KEGG pathway and gene ontology (GO) Biological Processes enrichment analysis identified pathways for DEGs between old versus young female mice seen only in the R1441C-Lrrk2 IFNγ-treated pMacs group. (E) Heat maps show KEGG Parkinson’s Disease pathway genes with significant old versus young DEGs seen only in R1441C-Lrrk2 IFNγ-treated pMacs. (F) KEGG pathway and GO BP enrichment analysis identified pathways for DEGs upregulated in IFNγ-treated pMacs from aged R1441C-Lrrk2 female but not male mice. For transcriptomic analysis, pMac-derived RNA was used from 4–6 mice per sex, genotype, treatment and age groups.

Figure 7.. R1441C-LRRK2 and Y1699C-LRRK2 monocyte-derived macrophages from PD patients exhibit hypophagocytosis and increased immune cell exhaustion.

(A, B) Cryopreserved PBMCs from three PD patients carrying either the R1441C-LRRK2 or Y1699C-LRRK2 mutation or from three non-manifesting mutation carriers (NMC) or nine healthy controls were thawed and plated in culture dishes in the presence of human M-CSF to differentiate myeloid cells into macrophages. Macrophages were incubated with 100μg of pHrodo E. coli and imaged every 20 minutes for 5 hours, and GFP⁺ cells (% of total) were quantified. Scale bars, 100μm. Three-way ANOVA, Bonferroni post hoc. Main effects of groups are reported in each graph key. (C-E) PD-L1⁺ cell counts and PD-L1 expression were quantified in total PBMCs by flow cytometry. Bars represent mean +/− SEM (n = 3–9 participants). One-way ANOVA, Bonferroni post hoc. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, ****= p < 0.001. (F) Monocyte-derived macrophages for the three groups were stained for PD-L1 and microscopy images were captured. Scale bars, 100μm. Representative images from one participant per group are shown.