Article Figures & Data
Figures
- Figure S1. GDNF family expression in mouse macrophages.
(A, B) mRNA expression of neurotrophins and their receptors in GM-CSF (A)– and M-CSF (B)–differentiated BMDMs by RT2 profiler array (n = 1). Genes with a fold change >2 are shown. (C, D, E) Relative mRNA expression (2−ΔCT × 104) of GFRα2, RET, and NRTN in GM-CSF BMDMs (C) (n = 5), M-CSF BMDMs (D) (n = 3) and mouse AMs (E) (n = 3). RT-qPCR data are normalised to the housekeeping genes RN18s and Hprt. (C, D, E) Data information: Data are shown as mean ± SEM (C, D, E).
Source data are available for this figure.
Source Data for Figure S1[LSA-2020-00780_SdataFS1.xlsx]
- Figure 1. Human lung macrophages express GFRα2 and induce RET expression after TLR activation, whereas lung epithelial cells express the ligand NRTN.
(A) Relative mRNA expression (2−ΔCT × 104) of GDNF family receptors and ligands in human lung macrophages (n = 3). (B) Relative mRNA expression of GFRα2, RET, and NRTN in peripheral blood monocytes (n = 3) or MDMs (n = 6) from non-matched donors. (C) Representative images of GFRα2 expression in human lung macrophages by immunofluorescence (GFRα2 or isotype control [red] DAPI [blue]). Scale bar = 20 μM. (D) Representative Western blot of GFRα2 and RET protein expression in MDMs (lanes 1–3) (n = 3), human lung macrophages (MΦs) (lanes 4–6) (n = 3) and PMA-treated THP-1 macrophage positive control (lane 7); β-actin housekeeping control. (E) mRNA expression of RET in MDMs after stimulation with IL-4, IL-10, IL-13 (all 20 ng/ml), polyI:C (10 μg/ml), LPS, or R848 (both 100 ng/ml) for 24 h (n = 3) expressed as a fold change over the average expression of untreated (UT) cells. (F) Relative mRNA expression (2−ΔCT × 104) of GDNF family receptors and ligands in A549 cells (n = 3). (G) ELISA analysis of NRTN in A549 cells stimulated with polyI:C (10 μg/ml), LPS, R848 (both 100 ng/ml), IFNα or IFNβ (both 20 ng/ml) for 24 h (n = 3). Data information: Data are shown as mean ± SEM from three to six independent experiments. (E, G) *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Tukey’s post hoc test with multiple comparisons (E, G).
Source data are available for this figure.
Source Data for Figure 1[LSA-2020-00780_SdataF1.xlsx]
- Figure S2. GDNF family expression in human bronchial epithelial cells and mouse influenza infection model.
(A, B) Relative mRNA expression (2−ΔCT × 104) of GDNF family ligands (A) and receptors (B) in BEAS-2B cells (n = 3). (C) ELISA analysis of NRTN in BAL fluid (BALF) from mice up to 42 days post infection with influenza virus (n = 4). Data information: Data are shown as mean ± SEM. (C) *P < 0.05, ***P < 0.001; one-way ANOVA with Tukey’s post hoc test with multiple comparisons (C).
Source data are available for this figure.
Source Data for Figure S2[LSA-2020-00780_SdataFS2.xlsx]
- Figure S3. PolyI:C induces type I IFN production by macrophages and expression of different RET isoforms.
(A, B) mRNA expression of IFNα (A) and IFNβ (B) in human MDMs stimulated with polyI:C (10 μg/ml), LPS (100 ng/ml), or R848 (100 ng/ml) for 24 h (n = 3). (C) mRNA expression of IFNα in human lung macrophages stimulated with polyI:C (10 μg/ml) for 24 h (n = 3). (D) GFRα2 mRNA expression in human lung macrophages stimulated with IFNα, IFNβ, IFNγ, or IFNλ (all at 20 ng/ml) for 24 h (n = 3). (E) mRNA expression of the RET isoforms, RET 51, and RET9, in human lung macrophages stimulated with IFNα or IFNβ for 24 h (n = 3). RT-qPCR data are normalised to the housekeeping genes RPLP0 or GAPDH and expressed as a fold change over the average expression of untreated (UT) cells. (F) Western blot of GFRα2 and RET in THP-1 monocytes from three separate experiments (lanes 1–3) (n = 3). Data information: Data are shown as mean ± SEM. (A, B, D) *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Tukey’s post hoc test with multiple comparisons (A, B, D).
Source data are available for this figure.
Source Data for Figure S3[LSA-2020-00780_SdataFS3.xlsx]
- Figure 2. Macrophage RET expression is indirectly stimulated by TLR agonists via type I IFNs.
(A, B, C) ELISA analysis of IFNβ release (n = 3) (A) and mRNA expression of IFNβ (B) or RET (C) (n = 6) in human lung macrophages stimulated with polyI:C (10 μg/ml) ± BX795 (5 μg/ml) for 24 h. (D, E, F) RET mRNA expression in human lung macrophages (D), human peripheral blood monocytes (E), and human MDMs (F) stimulated with IFNα, IFNβ, IFNγ, or IFNλ (all at 20 ng/ml) for 24 h (n = 3). (G, H, I) Representative Western blot (untreated [lane 1], 20 ng/ml IFNβ [lane 2], 100 ng/ml IFNβ [lane 3]) (G) and densitometry analysis of RET (H) and GFRα2 (I) protein expression relative to β-actin in human MDMs stimulated with 20 ng/ml or 100 ng/ml of IFNβ for 24 h (n = 3). (J) Representative images of RET expression in untreated or IFNβ-treated PMA-treated THP-1 macrophages (top panels; untreated [UT], bottom panels; IFNβ-treated [20 ng/ml], RET [red] DAPI [blue]). Scale bar = 40 μM. Data information: Data are shown as mean ± SEM from three to six independent experiments. (A, C, D, E, F, H) *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA with Tukey’s post hoc test with multiple comparisons (A, D, E, F, H); Kruskal–Wallis test with Dunn’s multiple comparisons test (C).
Source data are available for this figure.
Source Data for Figure 2[LSA-2020-00780_SdataF2.xlsx]
- Figure S4. The effect of NRTN on human lung macrophages and MMP9 production.
(A, B, C) mRNA expression of macrophage cell biology genes in human lung macrophages stimulated with NRTN (100 ng/ml) compared with untreated (A), IFNβ (20 ng/ml) compared with untreated (B) or NRTN + IFNβ compared with IFNβ alone (C) for 8 h by qPCR array (n = 1). qPCR array data are normalised to the average of five housekeeping genes and expressed as a fold change over untreated cells. (D, E) Representative Western blot and densitometry analysis of and MMP9 (E) in human lung macrophages stimulated with polyI:C (10 μg/ml), IFNβ (20 ng/ml) ± NRTN (100 ng/ml) for 24 h (n = 3). Densitometry analysis of MMP9 is expressed relative to β-actin. (F) ELISA analysis of MMP9 release from human lung macrophages stimulated with polyI:C (10 μg/ml) or IFNβ (20 ng/ml) ± NRTN (100 ng/ml) for 24 h (n = 3). Data information: Data are shown as mean ± SEM. (F) ***P < 0.001; Student’s unpaired two-tailed t test (F).
Source data are available for this figure.
Source Data for Figure S4[LSA-2020-00780_SdataFS4.xlsx]
- Figure 3. NRTN promotes MMP2 and MMP9 production from macrophages.
(A, B) Representative Western blot (A) and densitometry analysis (B) of MMP2 in human lung macrophages treated with polyI:C (10 μg/ml) or IFNβ (20 ng/ml) ± NRTN (100 ng/ml) for 24 h (n = 3). (C, D, E) Representative Western blot (C) and densitometry analysis of MMP2 (D) or MMP9 (E) protein expression in PMA-treated THP-1 macrophages stimulated with AD80 (0.5 μM) ± NRTN (100 ng/ml) for 24 h (n = 4). Densitometry analysis of MMP2 and MMP9 is expressed relative to β-actin. (F, G) Representative image (F) and quantification per cell and per cell area (G) of gelatin degradation by human lung macrophages stimulated with polyI:C (10 μg/ml) ± NRTN (100 ng/ml) for 24 h (n = 3). Each dot on the graph represents a field of view, minimum seven fields of view per donor. Scale bar = 50 μM. Data information: Data are shown as mean ± SEM from three or four independent experiments. (B, D, E, G) *P < 0.05, **P < 0.01, ***P < 0.001; Student’s paired two-tailed t test (B, D, E, G).
Source data are available for this figure.
Source Data for Figure 3[LSA-2020-00780_SdataF3.xlsx]
- Figure 4. NRTN reduces MMP1 and MMP3 production from macrophages.
(A, B) Representative Western blot (A) and densitometry analysis of MMP3 (B) in human lung macrophages stimulated with polyI:C (10 μg/ml) ± NRTN (100 ng/ml) for 24 h (n = 3). (C, D, E) Representative Western blot (C) and densitometry analysis of MMP1 (D) or MMP3 (E) protein expression in PMA-treated THP-1 macrophages stimulated with AD80 (0.5 μM) ± NRTN (100 ng/ml) for 24 h (n = 3–7). Densitometry analysis of MMP1 and MMP3 is expressed relative to β-actin. Data information: Data are shown as mean ± SEM from three independent experiments. (B, D, E) *P < 0.05; Student’s paired two-tailed t test (B, D, E).
Source data are available for this figure.
Source Data for Figure 4[LSA-2020-00780_SdataF4.xlsx]
- Figure 5. NRTN dampens the release of pro-inflammatory cytokines from human lung macrophages.
(A, B, C, D) ELISA analysis of IL-6 (A), IL-12p40 (B), TNFα (C) and IL-10 (D) release from human lung macrophages stimulated with polyI:C (10 μg/ml) or IFNβ (20 ng/ml) ± NRTN (100 ng/ml) for 24 h (n = 4). Data information: Data are shown as mean ± SEM from four independent experiments. (A, B) *P < 0.05, ***P < 0.001; one-way ANOVA with Tukey’s post hoc test with multiple comparisons (A, B).
Source data are available for this figure.
Source Data for Figure 5[LSA-2020-00780_SdataF5.xlsx]
- Figure 6. GDNF family expression is enhanced in macrophages from the lungs of chronic smokers and lung tumour tissue.
(A, B, C) Relative GFRα2 (A), RET (B), and MMP2 (C) mRNA expression (2−ΔCT × 104) in macrophages isolated from the lungs of patients categorised as “controls” (n = 16) or chronic obstructive pulmonary disease (GFRα2 and RET, n = 18; MMP2, n = 11). (D, E, F) Relative GFRα2 (D), RET (E), and MMP2 (F) mRNA expression in macrophages isolated from the lungs of patients categorised as non-smokers (n = 18) or smokers (GFRα2 and RET, n = 16; MMP2, n = 8). (G, H) Relative mRNA expression (2−ΔCT × 104) of GFRα2 (G) and RET (H) in macrophages isolated from lung tumour tissue and matching healthy margin tissue (n = 4). (A, B, C, D, E, F) Data information: Data are shown as median ± interquartile range (A, B, C, D, E, F). (D, F, H) *P < 0.05, **P < 0.01; Mann–Whitney U test (D, F), Student’s paired two-tailed t test (H).
Source data are available for this figure.
Source Data for Figure 6[LSA-2020-00780_SdataF6.xlsx]
- Figure 7. A novel role for the GDNF family on macrophages in respiratory viral infection.
TLR activation of airway epithelial cells enhances the production of NRTN, which binds to constitutively expressed GFRα2 on the surface of lung macrophages (1). Viral TLR activation on lung macrophages (2) triggers the release of IFNβ (3), which acts in an autocrine manner and binds to the IFNα/β receptor, IFNAR (4), to stimulate the production of RET at the mRNA and protein level (5). RET translocates to the cytoplasm and binds to GFRα2 (6), stimulating the production of MMP-2 (7) and dampening the pro-inflammatory cytokine response.
Supplementary Materials
In this Issue
Subjects
Related Articles
Cited By...
- No citing articles found.