In this paper, we show that macrophage activity associated with inflammation in the colon of IL-10−/− mice can be non-invasively detected and scored using 19F MRI utilizing a novel PFC tracer-imaging agent. Using immunofluorescence, qRT-PCR and in situ macrophage ablation, we demonstrate that PFC is localized within macrophages and that the cell quantity is reflected in the magnitude of the measured 19F signal in the colon. Furthermore, we show that the method is suitable to assay the effects of putative therapeutics used to treat colitis. The total imaging time for 19F MRI used in this work was ~8 min, which is practical for routine preclinical studies and testing of therapeutic test articles.
The non-invasive nature of 19
F MRI provides rapid assessment of the extent and 3D distribution of bowel inflammation in a manner that is not feasible by invasive procedures like biopsy or necropsy. Importantly, the extent of colon inflammation and age of onset are key factors that determine the progression of colitis into colitis-associated colon cancer.1
Because there is no endogenous MRI-detectable fluorine in the bowel, the only source of 19
F signal is inflammation-associated macrophage activity, and thus detection is unambiguous. The absence of background 19
F enables reliable lesion quantification, which is often challenging with various 1
H MRI modalities. In our study, it was difficult to assess inflammation by measuring bowel thickness by 1
H MRI alone, especially in descending colon where inflammation was mild. The 19
F signals were readily detected in the descending colon even when the inflammation was mild. Moreover, 3D reconstructions of bowel lesions are feasible without manual segmentation, offering views of the colon within the context of the whole abdominal cavity that provide an accurate picture of the extent and distribution of the diseased regions.
As a class of molecules, many PFCs are considered safe for in vivo
applications. Several PFCs have been used in human clinical trials as artificial oxygen carriers.32
The blood half-life of the PFC used in this study is ~12 h in mice. In tissues, PFC accumulates in the reticuloendothelial system initially and is slowly cleared via the lung. Recently, numerous studies have investigated the impact of PFC cell labeling on cellular phenotype and function in primary immune cells using a variety of sensitive in vitro
assays, eg, in the context of human dendritic cells30,33
and murine T cells.31,34,35
Cytotoxicity following PFC labeling was minimal, and there were no phenotypic alterations to the cells after labeling. These studies and others34,36–38
label cells with various PFC molecules in vitro
with the goal of performing in vivo
F cell-tracking studies after cell transfer to a subject, eg, to monitor T-cell migration in experimental models of diseases. In a prior study using PFC-labeled antigen-specific T cells, we reported extra-intestinal loci of inflammation in pancreas in a mucin1+
transgenic mouse model of IBD using 19
F NMR and MRI.34
We note that in the IL-10−/−
model utilized in this study there was no evidence of pancreatitis.
Quantitatively, in vitro
PFC emulsion uptake by phagocytic cells, such as fetal skin-derived dendritic cells, is empirically found to be on the order of ~5 × 1012
fluorine atoms per cell;30
for non-phagocytic cells, such as 9 l glioma cells and T cells, uptake ranges from approximately 2 × 1011
to 1 × 1012
fluorine atoms per cell.34,37
Assuming that macrophages uptake ~5 × 1012
atoms per cell in vivo
, we estimate that the magnitude of macrophages in the bowel wall is of order ~3 × 106
cells based on our measured 19
F levels in vivo
(). We note that the mean PFC uptake per macrophage in vivo
is not known and difficult to measure, unlike the ex vivo
labeling experiments. From immunofluorescence, it is estimated that 15–20% of macrophages in the colon contained PFC-DiI.
We demonstrated that ablation of macrophages in the circulation and abdominal organs with clodronate liposomes greatly reduces 19F signal in the colon. This result further demonstrates the selective labeling of macrophages by PFCs in vivo. Histological scoring demonstrated that macrophage ablation reduced inflammation in colon. This result indicates the importance of macrophages in the development of IBD.
We tested the prophylactic effects of two drugs, including dexamethasone and CsA, in IBD induction; the goal of these tests was to determine if 19
F MRI could be used to detect any putative therapeutic effects. Both drugs are used clinically to treat patients with IBD.39
In our model, dexamethasone increased inflammation, especially in the descending colon, whereas CsA had a small effect on the induction of colitis. We note that facilitation of colitis with dexamethasone was also observed in the acute phase of DSS-induced colitis,40,41
but it ameliorated colitis in the chronic phase of DSS-induced colitis.41
The exact mechanisms of these effects are currently not known. Differential effects of corticosteroids on the immune cells could partly explain the observed phenomenon.42
Neutrophils, the mediators of acute inflammation, are relatively resistant to corticosteroid action and may promote the initial acute inflammation phase in piroxicam-induced intestinal damage in our model which in turn could trigger uncontrolled inflammation in an immune-deficient model such as IL-10−/−
mice. Dexamethasone is also known to aggravate the adverse gastrointestinal effects, such as perforation and bleeding, when combined with non-steroidal, anti-inflammatory drugs such as piroxicam.43
CsA, on the other hand, reduced the severity of inflammation in the acute phase of DSS-mediated colitis by upregulating transforming growth factor-β
in the intestine. CsA reduces inflammation by suppressing the secretion of IL-2 by activated T cells.45
Activated CD4 +
T cells are the primary effectors that perpetuate colitis in IL-10−/−
mice that act synergistically with macrophages to promote inflammation. The initial induction phase of piroxicam-induced colitis in IL-10−/−
mice may be similar to acute phase of DSS-induced colitis.
In this study, we have employed a prophylactic IBD treatment model. Although, prophylactic treatment models can provide important insights into the mechanisms of disease, and in our study the basis of the 19F MRI signal generation, we acknowledge that it is less relevant clinically. In this work, our attempt was to establish a quantitative method of measuring bowel inflammation, and therapeutic interventions were primarily used for that purpose.
The MRI-active PFC molecule (perfluoropolyether) used in this study, designed for optimal MRI sensitivity (ie, large number of NMR-equivalent 19
F’s and short T1
ratio), has not yet been approved for clinical use. Related PFC molecules, such as perfluorooctlybromide, have been used as oxygen transport reagents in human clinical trials and have been used in MRI studies.46,47
However, perfluorooctyl-bromide is non-ideal for MRI, as it has a long T1
and displays multiple 19
F NMR peaks, which significantly compromises sensitivity as only one single peak can be used for imaging, and the other peaks must be suppressed using pre-excitation saturation pulses. Our view is that the results presented herein help provide a rationale for pursuing the development of optimal clinical MRI diagnostic reagents for IBD that are based on PFC emulsions. Moreover, the feasibility of performing 19
F MRI scans on a clinical scanner has been demonstrated in many studies38,48
with the addition of a dual-tuned 19
H radio-frequency coil.
Imaging using 18
and endomicroscopic-imaging approaches50
have also been used for quantifying bowel inflammation. 18
FDG imaging has been used in human studies. 19
F MRI may be a promising alternative to both of these techniques. 18
FDG PET measures metabolic activity and our approach measures macrophage content in the inflammatory sites. Both of these approaches are capable of measuring inflammatory activity. PET is sensitive but involves radioactive tracers, can have high background and lower spatial resolution. Moreover, it requires CT for anatomical localization. 19
F MRI does not use ionizing radiation, has better spatial resolution and 1
H and 19
F MRI can be performed on the same instrument to obtain complementary information. Endoscopic approaches are invasive, whereas 19
F MRI is capable of 3D visualization of bowel inflammation non-invasively.
Overall, macrophages have an important role in the pathogenesis of a large number of chronic inflammatory diseases and cancers. 19F MRI is capable of in vivo visualization and quantification of macrophage activity. We believe that in the future, 19F MRI technology will be an effective tool for monitoring macrophage activities in disease and for assessing treatment response.