MR imaging and targeting of a specific alveolar macrophage subpopulation in LPS-induced COPD animal model using antibody-conjugated magnetic nanoparticles

Abstract

Purpose: Targeting and noninvasive imaging of a specific alveolar macrophage subpopulation in the lung has revealed the importance for early and better diagnosis and therapy of chronic obstructive pulmonary disease (COPD). In this study, the in vivo effect of pulmonary administration of iron oxide nanoparticles on the polarization profile of macrophages was assessed, and a noninvasive free-breathing magnetic resonance imaging (MRI) protocol coupled with the use of biocompatible antibody-conjugated superparamagnetic iron oxide (SPIO) nanoparticles was developed to enable specific targeting and imaging of a particular macrophage subpopulation in lipopolysaccharide-induced COPD mice model.

Materials and methods: Enzyme-linked immunosorbent assay, Real-time polymerase chain reaction, and flow cytometry analysis were performed to assess the biocompatibility of PEGylated dextran-coated SPIO nanoparticles. Specific biomarkers for M1 and M2 macrophages subsets were selected for conjugation with magnetic nanoparticles. MRI protocol using ultra-short echo time sequence was optimized to enable simultaneous detection of inflammation progress in the lung and detection of macrophages subsets. Flow cytometry and immunohistochemistry analysis were finally performed to confirm MRI readouts and to characterize the polarization profile of targeted macrophages.

Results: The tested SPIO nanoparticles, under the current experimental conditions, were found to be biocompatible for lung administration in preclinical settings. Cluster of differentiation (CD)86- and CD206-conjugated magnetic nanoparticles enabled successful noninvasive detection of M1 and M2 macrophage subpopulations, respectively, and were found to co-localize with inflammatory regions induced by lipopolysaccharide challenge. No variation in the polarization profile of targeted macrophages was observed, even though a continuum switch in their polarization might occur. However, further confirmatory studies are required to conclusively establish this observation.

Conclusion: Coupling of magnetic iron oxide nanoparticles with a specific antibody targeted to a particular macrophage subpopulation could offer a promising strategy for an early and better diagnosis of pulmonary inflammatory diseases using noninvasive MRI.

Keywords: MRI; iron oxide nanoparticles; lipopolysaccharide; lung imaging; lung inflammation; macrophage tracking; magnetic resonance imaging.

Figure 1

Number of neutrophils, macrophages, lymphocytes, and total cells in BALF of (A) mice intrapulmonary exposed to LPS at different investigation time points, and (B) control, SPIO, LPS, and LPS-SPIO mice at 48 hours post-LPS challenge. Notes: Data expressed as mean ± standard deviation, n=6 per group. ^#, ^‡, *, P<0.05. Abbreviations: BALF, bronchoalveolar lavage fluid; LPS, lipopolysaccharide; SPIO, superparamagnetic iron oxide; h, hours.

Figure 2

Measurement of IL-12 and CXCL-10 levels as markers of M1 macrophages and IL-4 and CCL-22 levels as markers of M2 macrophages in BALF of control, SPIO, LPS, and LPS-SPIO groups at 48 hours post-LPS challenge. Notes: Data expressed as mean ± standard deviation, n=6 per group; *P<0.05. Abbreviations: BALF, bronchoalveolar lavage fluid; IL, interleukin; LPS, lipopolysaccharide; SPIO, superparamagnetic iron oxide; CXCL-10, chemokine (C-X-C motif) ligand 10; CCL-22, chemokine (C-C motif) ligand 22.

Figure 3

Relative gene expression of NOS-2, CXCL-10, and TNF as markers of M1 macrophages and CCL-17, CCL-22, Arg1, and IL-10 as markers of M2 macrophages in SPIO, LPS, and LPS-SPIO mice compared with control mice. GAPDH was analyzed as an internal control. Note: Data expressed as mean ± standard deviation, n=6 per group. Abbreviations: Arg, arginase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL, interleukin; LPS, lipopolysaccharide; NOS, nitric oxide synthase; SPIO, superparamagnetic iron oxide; TNF, tumor necrosis factor; CXCL-10, chemokine (C-X-C motif) ligand 10; CCL-17, chemokine (C-C motif) ligand 17.

Figure 4

Flow cytometry analysis of alveolar macrophages issued from the BALF of control, SPIO, LPS, and LPS-SPIO groups. (A) Representative histogram of CD86 (higher row) and CD206 (lower row) expression. (B) Percentage of cells expressing CD86, CD197, CD206, CD150, and CD11b. Notes: Error bars are standard deviation of triplicates. *P<0.05. Abbreviations: BALF, bronchoalveolar lavage fluid; LPS, lipopolysaccharide; SPIO, superparamagnetic iron oxide; CD, cluster of differentiation.

Figure 5

(A) Representative magnetic resonance images acquired on a 4.7T magnet, using a radial ultra-short echo time sequence of a control, LPS (48 hours post-LPS induction), SPIO, CD86-SPIO, and CD206-SPIO groups (2 hours post-intrapulmonary administration of either non-conjugated, CD86-conjugated, or CD206-conjugated SPIO nanoparticles) of 48 hours post-LPS-induced COPD in mice. Notes: (A) Red arrows highlight the presence of void signal dots related to the presence of the nanoparticles, which were found to co-localize with inflammatory regions with antibody-conjugation SPIO nanoparticles. (B) Quantification of hyperintensity pixels, averaged on a set of ten slices, with 0.5 mm inter-slices which were positioned at the same position in lung parenchyma of the different groups. Data expressed as mean ± standard deviation, n=6 per group. *P<0.05. Abbreviations: LPS, lipopolysaccharide; SPIO, superparamagnetic iron oxide; CD, cluster of differentiation.

Figure 6

Flow cytometry analysis of targeted macrophage subpopulations sorted using magnet attraction from total cellular extracts of 48 hours post-LPS-induced COPD mice lungs before and after intrapulmonary instillation of either CD86-conjugated (LPS + CD86-SPIO) or CD206-conjugated (LPS + CD206-SPIO) SPIO nanoparticles. (A) Representative histogram of CD86 (high row) and CD206 (lower row) expression. (B) Percentage of cells expressing CD8 and CD206 in the different groups. Notes: (+) and (−) refer to eluted bound or unbound cell fractions, respectively. Error bars are standard deviation of triplicates. *P<0.05. Abbreviations: COPD, chronic obstructive pulmonary disease; LPS, lipopolysaccharide; SPIO, superparamagnetic iron oxide; CD, cluster of differentiation.

Figure 7

Immunohistochemistry analysis of F4/80 (universal marker of macrophages), iNOS (marker for M1 macrophages), Arginase1 (marker for M2 macrophages), and Prussian blue (marker for iron oxide) staining in 48 hours post-LPS-induced COPD lung before and after intrapulmonary administration of either SPIO, CD86-SPIO, or CD206-SPIO nanoparticles. Note: Final original magnification, ×400. Abbreviations: COPD, chronic obstructive pulmonary disease; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; SPIO, superparamagnetic iron oxide; CD, cluster of differentiation.