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. 2018 Feb 8:9:210.
doi: 10.3389/fimmu.2018.00210. eCollection 2018.

Leucine-Rich Repeat Kinase 2 Controls the Ca2+/Nuclear Factor of Activated T Cells/IL-2 Pathway during Aspergillus Non-Canonical Autophagy in Dendritic Cells

Alicia Yoke Wei Wong  1   2 , Vasilis Oikonomou  3 , Giuseppe Paolicelli  3 , Antonella De Luca  3 , Marilena Pariano  3 , Jan Fric  1   4 , Hock Soon Tay  1 , Paola Ricciardi-Castagnoli  1   5 , Teresa Zelante  1   3
Affiliations

Leucine-Rich Repeat Kinase 2 Controls the Ca2+/Nuclear Factor of Activated T Cells/IL-2 Pathway during Aspergillus Non-Canonical Autophagy in Dendritic Cells

Alicia Yoke Wei Wong et al. Front Immunol. .

Abstract

The Parkinson's disease-associated protein, Leucine-rich repeat kinase 2 (LRRK2), a known negative regulator of nuclear factor of activated T cells (NFAT), is expressed in myeloid cells such as macrophages and dendritic cells (DCs) and is involved in the host immune response against pathogens. Since, the Ca2+/NFAT/IL-2 axis has been previously found to regulate DC response to the fungus Aspergillus, we have investigated the role played by the kinase LRRK2 during fungal infection. Mechanistically, we found that in the early stages of the non-canonical autophagic response of DCs to the germinated spores of Aspergillus, LRRK2 undergoes progressive degradation and regulates NFAT translocation from the cytoplasm to the nucleus. Our results shed new light on the complexity of the Ca2+/NFAT/IL-2 pathway, where LRRK2 plays a role in controlling the immune response of DCs to Aspergillus.

Keywords: Aspergillus; NRON; autophagy; dendritic cell; leucine-rich repeat kinase 2; nuclear factor of activated T cells.

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Figures

Figure 1
Figure 1
Leucine-rich repeat kinase 2 (LRRK2) is expressed in dendritic cells and is downregulated by Aspergillus fumigatus. (A) LRRK2 mRNA in D1 cells. Data is displayed as the mean gene expression ± SD of two biological replicates and normalized to the housekeeping protein, GAPDH. Differences found to be statistically significant by one-way ANOVA with Bonferonni’s Multiple Comparison post-test are indicated (NS, non-significant; ****, p < 0.0001). (B) Protein expression level of LRRK2 in whole cell lysates of D1 cells stimulated with A. fumigatus condia and swollen conidia. For densitometry analysis, band densities for LRRK2 were normalized to the band density of the housekeeping protein, GAPDH, after which the density of the LRRK2 band of the treated sample was compared with that of the untreated sample. Data is representative of three independent experiments. Abbreviations used: Untreated (Unt); A. fumigatus conidia (A-con); A. fumigatus swollen conidia (A-sw).
Figure 2
Figure 2
Leucine-rich repeat kinase 2 (LRRK2) is localized on lysosomes and endosomes of dendritic cells. (A) Western blot analysis of presence of TATA binding protein, Rab5, LRRK2, and LAMP-1 in a representative protein fraction obtained from the enrichment of lysosomes from D1 cells. Lysate from the protein fraction was run on both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and NuPAGE® to probe for the proteins indicated. Data are representative of three independent experiments (B–C), D1 cells were processed for immune-electron microscopy, stained for LAMP-1 or LRRK2, and labeled with appropriate secondary antibodies conjugated with 5 or 15 nm gold particles (size of particles indicated in superscript). Lysosomic and endosomic structures are as indicated. Electron micrographs of D1 cells show LAMP-1 positive lysosome (B), as well as endosomic structures positive for LRRK2 (C). Size of scale bars are as indicated.
Figure 3
Figure 3
Aspergillus fumigatus swollen conidia activate non-canonical autophagy and the formation of multivesicular bodies in dendritic cells. D1 cells were stimulated with A. fumigatus swollen conidia for 3 h. (A) Immunofluorescent staining of LC3 (red). Data is representative of two independent experiments. (B) Electron micrograph of Aspergillus cupping. The bottom panel is zoomed in from the area indicated in the top panel. Data is representative of two independent experiments. (C) Expression of LC3 and p62 proteins by western blot in whole cell lysates of D1 cells stimulated for 24 h with A. fumigatus swollen conidia. Densitometry for LC3 was performed on the LC3-II band to measure LC3-II turnover, while densitometry analysis of p62 was done on the upper band corresponding to its expected molecular weight of 60kD. Data is representative of two biological replicates. (D) Multilamellar bodies (encircled in yellow dashed lines) observed by electron microscopy in A. fumigatus swollen conidia-stimulated D1 cells and labeled for LAMP-1 or Leucine-rich repeat kinase 2 with 5 nm or 15 nm gold particles (size of particles indicated in superscript). Cells were incubated with fungi for 3 h. Size of scale bars are as indicated. Abbreviations: Untreated (Unt); A. fumigatus swollen conidia (A-sw).
Figure 4
Figure 4
Ca2+-NFAT-IL-2 axis is affected by early autophagic events, phagocytosis and LAP. nuclear factor of activated T cells translocation and IL-2 production of NFAT-luciferase reporter D1 cells that have been stimulated with A-sw for 6 h in the presence and absence of drugs inhibiting specific cellular processes. (A) Fungus-stimulated D1 cells in the presence and absence of the type III Phosphatidylinositol 3-kinase inhibitor 3MA, 10 mM, or the vacuolar H + ATPase inhibitor, Baf (50 nM), which inhibits the early and late stage of autophagy respectively. (B) Fungus-stimulated D1 cells in the presence or absence of the phagocytosis inhibitor, CytoD (2 µg/mL). (C) D1 cells stimulated with fungi in the presence or absence of the lysosomal acidification inhibiting combination of Leu/A. Data is displayed as the mean ± SD of five (in the case of Leu/A) or eight biological replicates. (D) Viability of D1 cells treated with fungus, drugs, or a combination of the both. (E) mRNA expression of IL-2 in D1 cells silenced for various mRNA regulating LAP in the presence or absence of A. fumigatus swollen conidia. Data is displayed as means ± SD of three biological replicates. Differences found to be statistically significant by one-way ANOVA with Bonferroni’s Multiple Comparison post-test are indicated (NS, non-significant; ***, p < 0.001; ****, p < 0.0001). Abbreviations: Untreated (Unt); A. fumigatus swollen conidia (A-sw); Vehicle non-treated control (NT); 3-methyladenine (3MA); Bafilomycin (Baf); Cytochalasin D (CytoD), combination of Leupeptin and ammonium chloride (Leu/A).
Figure 5
Figure 5
Leucine-rich repeat kinase 2 (LRRK2) deficiency in dendritic cells (DCs) leads to increased IL-2 production and nuclear factor of activated T cells (NFAT) translocation in response to Aspergillus fumigatus. (A) IL-2, IL-12/IL-23p40, and IL-23 cytokine production of wild-type (closed circles) and LRRK2−/− (open circles) bone marrow-derived DCs in response to fungal stimulation with A. fumigatus swollen conidia. Data is displayed as the mean cytokine concentration ± SD of two biological replicates and statistical significance determined by one-way ANOVA with Bonferroni’s Multiple Comparison post-test. (B) NFAT translocation measured by luminescence signal in A-sw-stimulated NFAT-luciferase reporter D1 cells upon LRRK2 silencing. Data is displayed as the mean luminescence signal ± SD of three biological replicates and statistical significance determined by Student’s t-test. Differences found to be statistically significant are indicated (*, p < 0.05; **, p < 0.01; ****, p < 0.0001). Abbreviations: Untreated (Unt); A. fumigatus swollen conidia (A-sw).
Figure 6
Figure 6
Regulation of IL-2 by NRON complex components. IL-2 cytokine production at 8 h post-exposure from A-sw stimulated D1 cells that were knocked down for NRON, sperm-associated antigen 9, PPP2R1A, or chromosome segregation 1-like through the use of shRNA-lentiviral particles. Data are displayed as the mean ± SD of three replicates. Differences between Aspergillus-stimulated cells found to be statistically significant by one-way ANOVA with Bonferroni’s Multiple Comparison post-test are indicated (NS, non-significant; ***, p < 0.001). Abbreviations: Untreated (Unt); A. fumigatus swollen conidia (A-sw).
Figure 7
Figure 7
Death-associated protein kinase 1 (DAPK1) expression during LAP is not controlled by Leucine-rich repeat kinase 2 (LRRK2). (A) Protein expression of DAPK1 in A. fumigatus swollen conidia-stimulated D1 cells. For densitometry analysis, band densities for DAPK1 were normalized to the band density of the housekeeping protein, β-actin, after which the density of the DAPK1 band of the treated sample was compared with that of the untreated sample. Data are representative of three independent experiments. (B) Immunofluorescent stanining of DAPK1 and brightfield microscopy of Aspergillus fumigatus swollen conidia-stimulated D1 cells. Signal intensity of the DAPK1 was quantified and displayed as mean fluorescence signal ± SD of three biological replicates. Brightfield imaging of the same cell is displayed with arrows indicating the presence of A. fumigatus swollen conidia in the cell. (C) Knockdown of LRRK2 in D1 cells was assessed by western blot. Data are represented as mean ± SD of three biological replicates and statistical significance determined by two way ANOVA (D) mRNA expression of DAPK1 in fungal-treated D1 cells that have been silenced for LRRK2. Data are displayed as the mean mRNA expression ± SD of three biological replicates and statistical significance determined by two way ANOVA. Differences found to be statistically significant are indicated (*, p < 0.05; **, p < 0.01). Abbreviations: A. fumigatus swollen conidia (A-sw); Untreated (Unt); D1 cells transduced with non-targeting control shRNA (Ctrl).
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