In vivo nanoparticle imaging of innate immune cells can serve as a marker of disease severity in a model of multiple sclerosis

Abstract

Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.

Keywords: EAE; MRI; USPIO; multiple sclerosis; nanoparticle imaging.

Fig. 1.

Cultured macrophages and microglia phagocytose CLIO-FITC. (A and B) Confocal images of bone marrow-derived macrophages (BMDMs) (A) and microglia (B) incubated with or without CLIO-FITC (500 µg/mL and 100 µg/mL, respectively). (C) Quantification of immunohistochemistry of CLIO uptake per macrophage subtype. (D) Representative flow cytometry histogram of CLIO uptake by macrophages. Numbers indicate the respective frequency of CLIO⁺ cells. (E) Quantification of flow cytometry data. (F) T2*-w MRI of macrophages incubated with CLIO-FITC. BMDMs (10⁵) were incubated for 2 h with or without CLIO. n = 3 independent biological replicates for C–F. (Scale bars: 50 µm for overview images in A and B; 5 µm for magnified Insets.)

Fig. 2.

In vivo nanoparticle uptake can be quantified by MRI. (A) Experimental outline. (B) EAE scores. (C and D) T2-w, T1-w after Gd, and T2*-w MRI before CLIO at onset of disease (C) and 2 d after disease onset (48 h after CLIO injection) (D). (E) Magnification of the cerebellum before (pre) (Top) and 48 h after CLIO administration (post) (Middle). (Bottom) The subtraction image (post CLIO minus pre CLIO). (F) Ex vivo T2* images 48 h after CLIO administration of a healthy (clinical score 0) and severely affected EAE animal (score 2.5. (G) Cerebellar volume with signal decrease on T2*-w images. (H) Correlation analysis of the volume of T2* signal decrease and the clinical score. (I) Correlation analysis of the volume of Gd enhancement and the clinical score. (Scale bars: 1 mm.)

Fig. S1.

Biodistribution and kinetics of the CLIO time course of T2*-w imaging following CLIO-FITC administration in a representative EAE animal. (A) Images were acquired before contrast administration (pre) and 15 min, 24 h, 48 h, and 72 h after (post) CLIO-FITC injection. (B) Spatial distribution of CLIO in a representative healthy mouse. MRI was performed before and 10 min and 48 h after CLIO administration. Coronal (Upper Row) and transverse (Lower Rows) T2-w images of the abdomen are shown. (C) Quantification of the CLIO signal intensity ratio (respective organ/muscle) in liver, spleen, blood (aorta), lymph node (inguinal lymph nodes, LN), and bone marrow (BM, thoracic vertebral body) before, 10 min after, and 48 h after CLIO. (D) Flow cytometry analysis of CLIO uptake in the respective organs in macrophages (CD11b⁺), T cells (CD3⁺), and B cells (CD19⁺) determined 48 h after CLIO administration compared with noninjected healthy control animals. (E) Confocal images of buffy coat white blood cells (after erythrocyte lysis) from animals with or without CLIO injection 48 h before imaging. n = 4 CLIO- injected mice, and n = 3 noninjected healthy controls. (Scale bars: 1 mm in A; 2 mm in B; 1 mm in lymph node Insets in B.)

Fig. 3.

Immunohistochemistry of immune cell infiltrates. (A) Confocal image (tile scan) of Iba-1⁺ macrophages/microglia in the cerebellum. CLIO-FITC was administered 48 h before. (B and C) Quantification of Iba-1⁺ cells (B) and CLIO-FITC uptake (C) in the cerebellar GM and WM (n = 7 mice for the acute EAE group and n = 3 mice for the healthy control group). a.u., arbitrary units; f.i., fluorescence intensity. (D and E) Confocal images of B cells (CD45R) (D) and T cells (CD3) (E) in EAE and healthy controls. (F–I) MRI (F and G) and immunohistochemistry (H and I) of cortical and midbrain lesions. (J) Staining for the inflammatory enzyme MPO. Arrowheads indicate MPO⁺ cells. (K) Representative section of a FluorClearBABB-cleared EAE brain and a healthy control brain 48 h after CLIO injection, acquired by UM. Brain clearing was performed in three EAE mice and three CLIO-injected healthy controls. (Magnification: K, 1×.) Confocal images were recorded as tile scans (composite image). (Scale bars: 500 µm for the overview image in A, Upper; 100 µm for the Inset in the overview in A; 20 µm for the magnified images in A, Lower; 20 µm for overview images in D and E; 5 µm for Insets In D and E; 1 mm in F, G, and K; 50 µm in H and I; 500 µm for the overview image in J; 100 µm for the magnified image in J.)

Fig. S2.

Assessment of axonal pathology and demyelination. (A) Experimental outline and Gd-enhanced MRI to assess BBB-D. (B) Confocal micrograph of an Iba-1–stained EAE animal 2 h after CLIO injection. Iba-1⁺ cells are negative for CLIO at this early time point but extend their processes close to CLIO particles (arrowhead in B, ii). (C) CLIO outlines vessels (arrowhead in C, 1) and is partly found free in the parenchyma (arrowheads in C, 2). (D) CD31⁺ endothelial cells do not take up CLIO when assessed 48 h after CLIO administration. (E) Assessment of axonal integrity by NFP staining in a healthy control and in an EAE animal. (F) Myelin staining by Luxol Fast Blue (LFB) in a representative EAE animal and in a healthy control. (Scale bars: 1 mm in A; 50 µm in B–D; 5 µm in B, ii; 500 µm in E and F.) n = 3 mice for each experiment.

Fig. S3.

MRI and immunohistochemistry of healthy control animals. (A and B) PTx-injected control animals do not show pathological changes on brain MRI. (C) Immunohistochemistry for Iba-1 and MPO, 48 h after CLIO injection. Control animals did not receive PLP but were injected with Ptx to rule out unspecific Ptx effects (n = 2 mice). Confocal images were recorded as tile scans (composite image). (Scale bars: 1 mm in A and B; 500 μm in C.)

Fig. S4.

Immunohistochemistry of EAE animals in the remission phase. (A) Representative immunohistochemical Iba-1 staining of an EAE animal in the remission phase 12 d after disease onset. CLIO-FITC was injected 2 d after disease onset, and CLIO-TAMRA was injected at day 10 after disease onset to investigate the kinetics of inflammatory cell influx. (B) Immunohistochemical Iba-1 staining of an EAE animal in the remission phase. CLIO-FITC was injected in the acute phase of the disease. (C) Quantification of Iba-1⁺ cells and CLIO-FITC uptake in the cerebellar GM and WM of EAE animals in the remission phase (n = 3 mice). The dashed line indicates the mean of healthy control mice. Confocal image in b was recorded as tile scan (composite image). (Scale bars: 50 μm in A; 500 μm in B; 100 μm in Inset.)

Fig. 4.

CLIO uptake of innate immune cells correlates with clinical severity. (A and B) Representative FACS plots for healthy control (A) and EAE (B) animals at onset + 2. Gating strategies are shown for the identification of the respective leukocyte subsets. (C) Histogram of CLIO⁺ cells for each cell population. (D and E) Flow cytometry quantification in the cerebellum (D) and cerebrum (E). (F and G) Percentage of CLIO⁺ cells. (H) Spearman correlation analysis of cerebellar macrophage frequency and EAE score. (I) Correlation analysis of CLIO⁺ cerebellar macrophage frequency and EAE score. Dots in D–I indicate single animals. n = 4 mice for healthy controls and n = 5–19 mice for EAE groups. Flow cytometry data are pooled from three independent experiments.

Fig. S5.

Gating strategy for the isolation of lymphocytes and myeloid cells. (A and B) Representative flow cytometry plots of flow analysis, illustrating the gating strategy for the lymphocyte (A) and myeloid (B) subsets. (C–E) CLIO uptake for the respective cell type in the cerebellum and spinal cord. Mϕ, macrophage. The statistical analysis in C was performed with one-way ANOVA and Tukey’s correction for multiple comparisons. n = 5 mice.

Fig. 5.

Ferumoxytol imaging delineates EAE lesions. (A and B) T2-w and T2*-w images at disease onset before ferumoxytol injection (A) and 1 d after EAE onset (24 h post ferumoxytol injection) (B). (C) Subtraction image (post ferumoxytol minus pre ferumoxytol) demonstrates signal loss in areas of ferumoxytol deposition (arrowheads). (D) Cerebellar volume with signal decrease on T2*-w images pre and post ferumoxytol administration. (E) Micrographs of DAB-enhanced Prussian blue staining for iron. CLIO section is shown as a positive control. n = 4 mice. (Scale bars: 1 mm in A–C; 20 µm in E.)