Preferential Macrophage Recruitment and Polarization in LPS-Induced Animal Model for COPD: Noninvasive Tracking Using MRI

<div><p>Noninvasive imaging of macrophages activity has raised increasing interest for diagnosis of chronic obstructive respiratory diseases (COPD), which make them attractive vehicles to deliver contrast agents for diagnostic or drugs for therapeutic purposes. This study was designed to monitor and evaluate the migration of differently polarized M1 and M2 iron labeled macrophage subsets to the lung of a LPS-induced COPD animal model and to assess their polarization state once they have reached the inflammatory sites in the lung after intravenous injection. <i>Ex vivo</i> polarized bone marrow derived M1 or M2 macrophages were first efficiently and safely labeled with amine-modified PEGylated dextran-coated SPIO nanoparticles and without altering their polarization profile. Their biodistribution in abdominal organs and their homing to the site of inflammation in the lung was tracked for the first time using a free-breathing non-invasive MR imaging protocol on a 4.7T magnet after their intravenous administration. This imaging protocol was optimized to allow both detection of iron labeled macrophages and visualization of inflammation in the lung. M1 and M2 macrophages were successfully detected in the lung starting from 2 hours post injection with no variation in their migration profile. Quantification of cytokines release, analysis of surface membrane expression using flow cytometry and immunohistochemistry investigations confirmed the successful recruitment of injected iron labeled macrophages in the lung of COPD mice and revealed that even with a continuum switch in the polarization profile of M1 and M2 macrophages during the time course of inflammation a balanced number of macrophage subsets predominate.</p></div

Examples of Immunohistochemical (IHC) staining of lungs sections of ctrl/ctrl, COPD/ctrl, COPD/M1 and COPD/M2 groups at 2 hours post-injection of macrophages using: F4/80 (first row) as universal marker for macrophages, iNOS (second row) and Arginase1 (third row) as marker for M1 and M2 macrophages respectively and of Prussian blue iron staining (fourth row) in adjacent sections revealing the presence of iron oxide nanoparticles (blue dots) in macrophages injected groups.

<p>Scale bar: 100 µm.</p

Flow cytometry analysis of alveolar macrophages in control/control, COPD/Control (48 h post LPS intrapulmonary exposition), COPD/M1 and COPD/M2 (2 h post-injection of iron-labeled macrophages) groups for both retained (i.e., iron loaded) and retained fractions.

<p>a- representative histogram of CD86 expression (higher row) and CD206 (lower row). b- Surface membrane receptor expression percentage of CD86, CD197, CD206 and CD150. Error bars are standard deviation of triplicates. *p<0.05.</p

Zeta potential of amine-modified PEGylated dextran-coated iron oxide nanoparticles assessed in ultrapure water at 25°C.

<p>Zeta potential of amine-modified PEGylated dextran-coated iron oxide nanoparticles assessed in ultrapure water at 25°C.</p

MR Images acquired using ultra-short echo time (UTE) sequence of control and LPS-induced COPD lungs, with or without injection of M2 macrophages (a).

<p>From top to bottom: Control, Control/M2, COPD, COPD/M2 groups imaged at (from left to right) −10 min, 2 h, 24 h and 168 h post M2 macrophages injection. Black arrows highlight the inflammatory regions in COPD groups and red arrows highlight the presence of void signal dots related to higher macrophage infiltrations in the inflammatory lungs. Signal-to-noise (SNR) attenuation of lung parenchyma, during the 7 days follow-up study, measured before and after intravenous injection of either free SPIO, SPIO labeled M1 or M2 macrophages in control and COPD mice (b). Error bars are standard deviation of triplicates. Representative regions of interest (ROI) which were drawn around apparent vascular structures (filled in red) and subtracted from the map to retain lung parenchyma (c).</p

Relative percentage of viability and reactive oxygen species generation of M1 and M2 SPIO labeled macrophages compared to unlabeled macrophages subsets after the overnight chase period (a).

<p>Nitric oxide (NO) release as marker of iNOS activity (left) in M1 macrophages and Arginine-derived urea production (right) as marker of Arginase1 activity in M2 macrophages (b). Error bars are standard deviation of triplicates.</p

Interleukins (IL-12, IL-4) and Chemokines (CCL-22 and CXCL-10) levels quantified by ELISA assay obtained from BAL samples of ctrl/ctrl, COPD/ctrl, COPD/M1 and COPD/M2 animal groups at 2 h post macrophage injection.

<p>Data expressed as mean ± SD, n = 5 per group. *p<0.05; **p<0.01.</p

MR images acquired using susceptibility-weighted gradient echo sequence showing the liver (upper row) and the spleen and kidneys (lower row) pre-injection (−1 h) and at 2 hours and 7 days post-injection of either free SPIO or SPIO labeled M2 macrophages in control mice groups (a).

<p>Contrast-to-noise (CNR) variation during the 7 days follow-up study for the spleen (left side) and the liver (right side) before and after intravenous injection of either free SPIO, SPIO labeled M1 or M2 macrophages in control and COPD animal groups (b). Error bars are standard deviation of triplicates.</p

Th17 cytokines induce pro-fibrotic cytokines release from human eosinophils

International audienceBackgroundSubepithelial fibrosis is one of the most critical structural changes affecting bronchial airway function during asthma. Eosinophils have been shown to contribute to the production of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this process is not fully understood.ObjectiveIn this report, we investigated whether cytokines associated with inflammation during asthma may induce eosinophils to produce pro-fibrotic cytokines.MethodsEosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production of TGF-β and IL-11 was determined using real time PCR and ELISA assays.ResultsThe basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2 cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.ConclusionsTh17 cytokines might contribute to airway fibrosis during asthma by enhancing production of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such as allergic and autoimmune diseases

Activation of p38 MAP kinase is required for Th-17 cytokines migratory effect on B cells.

<p>(A) B cells isolated from asthmatic patients were pre-treated with different kinase inhibitors or DMSO carrier for 1 hr before being incubated with Th-17 cytokines in a migration assay. Data is presented as % migration of B cells relative to negative control (medium). (n = 6) (B) Western blots showing p38 MAPK phosphorylation in B cells following Th-17 cytokine stimulation. Asthmatic B cells (2×10⁶) stimulated with Th-17 cytokines were lysed using 1x RIPA buffer and cell lysates were resolved using western analysis. Blots were probed using anti-phospho-p38 and anti-p38 MAPK antibodies. (C) Ratio of p-p38 over p38 band intensity were determined using densitometer and data were presented as fold increase in p38 phosphorylation ratio following Th-17 cytokine stimulation compared to non-stimulated condition (medium). Data is expressed as means ± SD (n = 5). *P<0.05 is considered significant.</p