Original Research n Molecular
Imaging
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Multiple Sclerosis: Myeloperoxidase Immunoradiology Improves Detection of Acute and Chronic Disease in Experimental Model1 Benjamin Pulli, MD Lionel Bure, MD Gregory R. Wojtkiewicz, MSc Yoshiko Iwamoto, BSc Muhammad Ali, MD Dan Li, MD Stefan Schob, MD Kevin Li-Chun Hsieh, MD2 Andreas H. Jacobs, MD John W. Chen, MD, PhD
Purpose:
To test if MPO-Gd, a gadolinium-based magnetic resonance (MR) imaging probe that is sensitive and specific for the proinflammatory and oxidative enzyme myeloperoxidase (MPO), which is secreted by certain inflammatory cells, is more sensitive than diethylenetriaminepentaacetic acid (DTPA)-Gd in revealing early subclinical and chronic disease activity in the brain in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis.
Materials and Methods:
The protocol for animal experiments was approved by the institutional animal care committee. A total of 61 female SJL mice were induced with EAE. Mice underwent MPOGd– or DTPA-Gd–enhanced MR imaging on days 6, 8, and 10 after induction, before clinical disease develops, and during chronic disease at remission and the first relapse. Brains were harvested at these time points for flow cytometric evaluation of immune cell subtypes and immunohistochemistry. Statistical analysis was performed, and P , .05 was considered to indicate a significant difference.
Results:
MPO-Gd helps detect earlier (5.2 vs 2.3 days before symptom onset, P = .004) and more (3.1 vs 0.3, P = .008) subclinical inflammatory lesions compared with DTPAGd, including in cases in which there was no evidence of overt blood-brain barrier (BBB) breakdown detected with DTPA-Gd enhancement. The number of MPO-Gd–enhancing lesions correlated with early infiltration of MPOsecreting monocytes and neutrophils into the brain (r = 0.91). MPO-Gd also helped detect more lesions during subclinical disease at remission (5.5 vs 1.3, P = .006) and at the first relapse (9.0 vs 2.7, P = .03) than DTPA-Gd, which also correlated well with the presence and accumulation of MPO-secreting inflammatory cells in the brain (r = 0.93).
Conclusion:
MPO-Gd specifically reveals lesions with inflammatory monocytes and neutrophils, which actively secrete MPO. These results demonstrate the feasibility of detection of subclinical inflammatory disease activity in vivo, which is different from overt BBB breakdown.
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From the Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, 185 Cambridge St, Boston, MA 02114 (B.P., L.B., G.R.W., Y.I., M.A., D.L., S.S., K.L.C.H., J.W.C.); Department of Radiology, Massachusetts General Hospital, Boston, Mass (B.P., J.W.C.); and European Institute for Molecular Imaging, University of Münster, Münster, Germany (A.H.J.). From the 2012 RSNA Annual Meeting. Received June 25, 2014; revision requested August 6; revision received August 11; accepted August 27; final version accepted September 19. B.P. supported by a fellowship from the Ernst Schering Foundation (Berlin, Germany) and a grant from the National Natural Science Foundation (Beijing, China). Address correspondence to J.W.C. (e-mail: [email protected]).
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Current address: Medical Imaging Center, Taipei Medical University Hospital, Taipei, Taiwan, Republic of China. 2
Online supplemental material is available for this article.
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M
ultiple sclerosis (MS) is the most common cause of disability in young adults (1). Although the pathogenesis of MS remains elusive, the presence of CD4+ autoreactive T-lymphocytes in the blood and brain of patients with MS suggest that it is a T-lymphocyte driven disease (2). Demyelination and axonal damage, however, are more likely caused by innate immune cells such as monocytes, macrophages, and neutrophils (2). Accordingly, in experimental autoimmune encephalomyelitis (EAE), the most commonly used experimental model of MS, depletion or inactivation of monocytes (3,4), microglia (5), and neutrophils (6) effectively suppresses disease. Early diagnosis of active MS with prompt treatment has been found to delay clinical relapses and decrease axonal loss. Magnetic resonance (MR) imaging is routinely used to assess disease
Advances in Knowledge nn Molecular MR imaging of myeloperoxidase (MPO) helps detect inflammatory lesions in experimental autoimmune encephalomyelitis (EAE) earlier (5.2 vs 2.3 days before symptom onset, P = .004), when there is no overt evidence of blood-brain barrier (BBB) breakdown (as detected by means of conventional gadolinium enhancement) or edema and/or demyelination (as detected with T2-weighted imaging).
activity in patients with MS but underreports inflammatory lesions, especially in the cortex (7). Moreover, only weak correlation has been found between MR imaging lesion volume and disease progression to disability (8,9). Gadoliniumenhancing lesions reflect blood-brain barrier (BBB) breakdown rather than active inflammation and do not predict relapse rate (10). This clinical-radiologic paradox might be explained by inflammatory activity behind an intact or partially repaired BBB (11,12). Myeloperoxidase (MPO) is an important oxidative enzyme secreted by innate immune cells (13). A higher-expressing MPO phenotype has been associated with early-onset MS (14), and MPO expression has been detected in white (15) and gray (16) matter plaques in patients with MS. The MPO-targeted MR imaging probe MPO-Gd (17,18) has been used to demonstrate MPO activity in vivo in EAE (19) and has been shown to be sensitive to treatment with a preclinical MPO inhibitor (20). However, it remains unclear if MPO-Gd can help detect acute and chronic subclinical inflammation, when the BBB is mostly closed, with higher sensitivity than diethylenetriaminepentaacetic acid (DTPA)-Gd. The aim of this study was to test if MPO-Gd is more sensitive than DTPAGd in the detection of early subclinical and chronic disease activity in the brain in EAE, a mouse model of MS.
nn MPO imaging also helps detect more lesions in disease remission (5.5 vs 1.3, P = .006), when disease is thought to be silent, and at the first relapse (9.0 vs 2.7, P = .03). nn MPO imaging results correlate well (r = 0.91 for subclinical acute and r = 0.93 for chronic EAE) with the presence of MPOsecreting proinflammatory monocytes and neutrophils in the brain, which migrate into the brain before the onset of clinical symptoms and remain active in disease remission. Radiology: Volume 275: Number 2—May 2015 n radiology.rsna.org
Implications for Patient Care nn On translation, MPO imaging could allow specific detection of the proinflammatory disease burden in multiple sclerosis (MS), extending on information that conventional T2-weighted imaging (edema/demyelination), gadolinium (BBB breakdown), and iron oxide (infiltration of phagocytic cells that can cause damage or repair) provide. nn Because persistent inflammation during clinical and radiologic remission correlates with disease progression in MS, MPO imaging may help predict progression to disability.
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Materials and Methods EAE Induction The protocol for animal experiments was approved by the institutional animal care committee. EAE was induced in 61 female SJL mice that were 6–10 weeks old (B.P., with 4 years of experience in EAE induction) with synthetic proteolipid protein (PLP139–151; NeoBioscience, Cambridge, Mass), as previously described (19,20). Briefly, each mouse was injected subcutaneously with a suspension containing 400 mg Mycobacterium tuberculosis H37RA (Difco) and 100 mg PLP139–151 in one part complete Freund adjuvant (CFA; Sigma, St Louis, Mo) mixed with one part phosphate-buffered saline. Within 2 hours of induction and on day 2, mice received 0.1 mg pertussis toxin (Sigma) intravenously. Sham-protocol mice were induced with identical steps, except that PLP was not used. With this induction protocol, disease onset was typically around days 11–13,
Published online before print 10.1148/radiol.14141495 Content codes: Radiology 2015; 275:480–489 Abbreviations: BBB = blood-brain barrier DTPA = diethylenetriaminepentaacetic acid EAE = experimental autoimmune encephalomyelitis MPO = myeloperoxidase MS = multiple sclerosis USPIO = ultrasmall superparamagnetic iron oxide Author contributions: Guarantors of integrity of entire study, B.P., G.R.W., S.S., J.W.C.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; agrees to ensure any questions related to the work are appropriately resolved, all authors; literature research, B.P., L.B., G.R.W., S.S., K.L.C.H., J.W.C.; experimental studies, B.P., L.B., G.R.W., Y.I., M.A., D.L., S.S., K.L.C.H., J.W.C.; statistical analysis, B.P., L.B., G.R.W.; and manuscript editing, B.P., G.R.W., M.A., S.S., K.L.C.H., A.H.J., J.W.C. Funding: This research was supported by the National Institutes of Health (grants R01-NS070835 and R01-NS072167). Conflicts of interest are listed at the end of this article. See also Science to Practice in this issue.
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with a first relapse occurring around days 24–30 after induction. The beginning of our study was induction with EAE, and mice were sacrificed either on days 6, 8, or 10 after induction (early subclinical disease), at remission (days 20–25 after induction), or at the first relapse (days 24–30 after induction.
Clinical Scoring Mice were checked daily for any signs of EAE. Weights were recorded daily. Clinical assessment for disease activity was performed by using the standard five-point staging system for EAE with the following scale (B.P. and D.L., who were blinded to which imaging agent was used): score 0, no signs; score 1, complete tail limpness without limb weakness; score 2, partial limb weakness and impaired righting reflex; score 3, at least one limb with complete paralysis; score 4, complete paralysis; and score 5, moribund. MPO-Gd Molecular MR Imaging To measure the specific MPO activity within the inflamed brain in vivo in living animals, we performed serial MPO-Gd (bis-5-hydroxytryptamide-diethylenetriaminepentaacetate gadolinium) molecular MR imaging in EAE mice (19,20). Early, subclinical lesions were detected on day 6, 8 and 10, when mice were asymptomatic. Lesions in remission and those in the first relapse were detected between days 18–23 and days 24–30, respectively, depending on the clinical score. Mice were considered to be in remission when the clinical score improved more than 1 point to either 0 or 1. A relapse was defined as weight loss of at least 0.5 g and an increase in the clinical score of at least 1 (Fig E1, A [online]). MPO-Gd is an activatable MR imaging agent that reports extracellular MPO activity in vivo with high specificity and sensitivity (17,18). MPO-Gd was synthesized as previously described (17–19). MR imaging was performed by using an animal 4.7-T MR imaging unit (Bruker, Billerica, Mass) consisting of mouse coronal T2-weighted images and T1-weighted images obtained before and after intravenous administration of MPO-Gd (0.3 mmol per kilogram of 482
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body weight) or DTPA-Gd (0.3 mmol/ kg). Mice were randomized to undergo imaging with either MPO-Gd or DTPAGd, as seen in the imaging flowchart (Fig E1b [online]). MR images were independently evaluated by two authors (B.P. and L.B., with 4 and 3 years of experience in interpreting MR images, respectively), who were blinded to the imaging agent used, the clinical scores of the mice, and the sham versus experimental group. Total lesion volume, total lesion count, and mean lesion volume were calculated.
cell numbers of different cell populations were calculated as total cells multiplied by the percentage within the respective cell population gate. Data were acquired with a flow cytometer (LSR II; BD Biosciences) and were analyzed with dedicated software (FlowJo 887; Tree Star, Ashland, Ore). Flow cytometric analysis was performed by one author (B.P., with 4 years of experience in flow cytometry of brain leukocytes). To evaluate the inflammatory cell infiltration specific to EAE, mice with EAE were compared with sham-protocol mice.
Isolation of Brain Inflammatory Cells and Flow Cytometry Mice were transcardially perfused with 20 mL ice-cold phosphate-buffered saline. Brain leukocytes were extracted by density centrifugation over a discontinuous Percoll gradient, as previously described (21). Briefly, brain tissue was mechanically ground in a glass tissue homogenizer in 8 mL of 30% Percoll solution (GE Healthcare, Boston, Mass), filtered through a 40-mm cell strainer (BD Biosciences, San Jose, Calif), and overlaid over 3 mL 70% Percoll. The density gradient was centrifuged at 650 g for 25 minutes at 18°C. After centrifugation, a thick myelin-containing layer at the top was removed, and the brain leukocytes at the 30/70 interface were collected. The cells were then counted and stained for flow cytometry. All antibodies were purchased from BD Bioscience unless otherwise indicated. The following antibodies were used: anti-CD90, 53–2.1; anti-NK1.1, PK136; anti-B220, RA3–6B2; anti-CD49b, DX5; anti-Ly-6G, IA8; anti-TER-119, TER-119; anti-CD11b, M1/70; anti-Ly-6C, AL-21; and anti-F4/80, C1:A3–1 (BioLegend, San Diego, Calif). For intracellular staining of MPO, cells were fixed and permeabilized (Cytofix/Cytoperm; BD Bioscience) after staining for cell surface markers; this was followed by staining for MPO using anti-MPO (clone 8F4; Hycult Biotech, Plymouth Meeting, Pa). When appropriate, a streptavidin-conjugated secondary antibody (eBioscience, San Diego, Calif) was used to label biotinylated antibodies. Our gating strategy can be seen in Figure E2 (online). The
Histopathologic Analysis Histopathologic analysis was performed to assess for MPO-expressing inflammatory cells. Fresh-frozen 5-mm brain sections were incubated in 1% hydrogen peroxide solution for 10 minutes and were blocked with 4% normal rabbit or goat serum for 30 minutes at room temperature. The sections were incubated with anti-MPO (Ab-1; Thermo Fisher Scientific, Waltham, Mass). A biotinylated secondary antibody (Vector Laboratories, Burlingame, Mass) was used. Statistical Analysis Results are reported as means 6 standard deviations. The data were tested for normality by using the D’AgostinoPearson normality test. If normality was not rejected, the t test was used. Otherwise, we used the nonparametric Mann-Whitney U test. The Fisher exact test was used to compare the number of mice found to have positive MR imaging findings with MPO-Gd versus DTPA-Gd. The Pearson correlation coefficient was calculated for MPO-Gd lesion counts versus number of MPOexpressing cells at flow cytometry. P , .05 was considered to indicate a significant difference. We used software for statistical analysis (GraphPad Prism 5; GraphPad Software). Results Detection of Early, Subclinical Disease with MPO-Gd MR Imaging MPO-Gd–enhanced molecular MR imaging helped detect at least one
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inflammatory lesion on day 8.0 6 1.1, while DTPA-Gd enhanced MR imaging was first positive on day 9.7 6 0.8 (P = .008). MPO-Gd first showed lesions 5.3 days 6 1.3 versus 2.3 days 6 1.0 before the onset of clinical symptoms (P = .004, Fig 1). On day 8, MPO-Gd showed significantly more lesions than DTPA-Gd (3.1 6 2.0 vs 0.3 6 0.8, P = .008). We also found that the average lesion size at the time of first detection was much smaller with MPO-Gd (4.6 mm3 6 3.1)
than with DTPA-Gd (38.5 6 31 mm3, P = .009, Fig 1). With MPO-Gd, 13% (one of eight) of mice were found to have a positive MR imaging result on day 6; by day 8, this number increased to 88% (seven of eight). With DTPA-Gd, no lesions were found on day 6, and only one (17%) of six mice was found to have a lesion on day 8 (P = .026 for day 8 comparison between MPO-Gd and DTPAGd). By day 10, lesions were found in all mice, regardless of imaging agent (Fig
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1). To evaluate if MPO-Gd enhancement also preceded T2 signal changes, which can be suggestive of edema and/or demyelination, we compared images from mice imaged with MPO-Gd on day 8. All lesions detected on day 8 with MPO-Gd were not seen on T2-weighted MR images, and no other lesions were detected (3.1 6 2.0 vs 0.0 6 0.0 lesions for MPOGd vs T2, P = .002, Fig 1). Representative images from mice imaged on day 8 with MPO-Gd and
Figure 1
Figure 1: Earliest detection of inflammation with MPO-Gd MR imaging in subclinical EAE. Molecular MR imaging with MPO-Gd (upper row) and DTPA-Gd (lower row) on day 6 (left) and day 8 (right) after disease induction reveals a small inflammatory lesion visible with MPO-Gd but not with DTPA-Gd (arrow). The inflammatory lesion seen with MPO-Gd was determined to correspond to an area of MPO-positive cells at immunohistochemistry, while no other areas containing MPO-positive cells were seen. MR image quantification revealed that MPO-Gd helps detect lesions earlier (more days before symptom onset, and with a higher percentage of positive MR imaging findings on postinduction day 8) and helps detect more and smaller lesions than DTPA-Gd. Day of disease onset was similar in the two groups. MPO-Gd also revealed more subclinical lesions than T2-weighted imaging (n = 8 for MPO-Gd and T2-weighted imaging, n = 6 for DTPA-Gd). ∗ = P , .05, ∗∗ = P , .01. p.i. = Postinduction. All data are means 6 standard errors of measurement. Radiology: Volume 275: Number 2—May 2015 n radiology.rsna.org
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Figure 2
Figure 2: Subclinical, early inflammatory lesions detected with MPO-Gd MR imaging on day 8 after EAE induction. A, Images show inflammatory lesions (arrows) in mice imaged with MPO-Gd (top row) and the absence of overt BBB breakdown as depicted by DTPA-Gd (bottom row). B, Inflammatory lesions (arrows) in mice imaged with MPO-Gd are detected on postcontrast T1-weighted (top) but not precontrast T2-weighted images (bottom).
DTPA-Gd can be seen in Figure 2, A. Postcontrast images and T2-weighted images from mice imaged on day 8 are shown in Figure 2, B. To confirm the specificity of MPO-Gd, we performed immunohistochemistry for MPO, where we could confirm a lesion with MPOpositive cells at a location corresponding to the MR imaging results (Fig 1). In line with a successful randomization, we did not find any difference in the clinical disease severity, as shown by similar clinical score profiles (Fig E3, A [online]), disease onset (P = .50, Fig 1), and cumulative disease scores (P = .55, Fig E3, B [online]).
Inflammatory Cell Subsets in Subacute, Early EAE In line with MPO-Gd imaging findings that in 88% of mice at least one discernible lesion was detectable as early as day 8, we found a significant increase in MPO-positive leukocytes with flow cytometry on day 8 compared with those in sham-protocol mice (P = .035). Similar results were observed on day 10 484
(P = .001, Fig 3, A). However, there was no significant increase in MPO-expressing leukocytes on day 6 (P = .16), consistent with imaging results. Next, we identified the leukocyte subtypes expressing MPO. Fifty-five percent were neutrophils and 38% were Ly-6Chigh monocytes, while neither microglia (3%) nor lymphocytes (1%) contributed significantly (Fig 3, A). In line with these findings, we detected a significant increase in Ly-6Chigh monocytes on days 8 and 10 compared with those in shamprotocol mice (P = .002 for day 8 and P , .001 for day 10; Fig 3, B). A similar trend was found for neutrophils, which were also significantly increased on day 8 and 10 compared with those in shamprotocol mice (P , .029 for day 8 and P , .001 for day 10; Fig 3, B). Last, we measured the MPO content of these different cell subsets. Neutrophils were found to have the most MPO (median fluorescent intensity [MFI], 3785; P = .002), followed by Ly-6Chigh monocytes (MFI, 2069; P = .002). Microglia (MFI, 771; P = .28) and lymphocytes (MFI,
314; P = .87) were not found to express MPO (isotype control MFI, 317; Fig E4, A [online]). In the blood, neutrophils (MFI, 4252; P = .023) and Ly-6Chigh monocytes (MFI, 3303; P = .023) were found to express MPO, while Ly-6Clow monocytes did not (MFI, 1627; P = .12) compared with isotype control (MFI, 1063; Fig E4, B [online]). Finally, the number of MPO-positive cells on days 6, 8, and 10 correlated well with MPO-Gd MR imaging lesion count (r = 0.91, P = .001; Fig E5, A [online]). Cumulatively, these results form a biologic basis for our imaging findings: MPO-secreting neutrophils and Ly-6Chigh inflammatory monocytes infiltrate as early as day 8 after induction, coinciding with the time of earliest detection of inflammation at MPO-Gd MR imaging.
Detection of Chronic Disease Activity with MPO-Gd MR Imaging At remission, we found a significantly higher number of lesions with MPO-Gd than with DTPA-Gd (5.5 6 0.8 vs 1.3 6 1.0 lesions per mouse, P = .006, Fig 4).
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Figure 3
Figure 3: Inflammatory cell subsets in subacute, early EAE. A, MPO-expressing cells accumulate in the brain on days 8 and 10 after induction. About 55% of MPO-expressing cells are neutrophils (red), and 45% are inflammatory (Ly-6Chigh) monocytes (blue). Microglia (black) and lymphocytes do not significantly contribute to MPO-expressing cells (n = 6 per group). B, Inflammatory (Ly-6Chigh) monocytes (blue) and neutrophils (red) infiltrate the brain on days 8 and 10 after induction (n = 6 per group). ∗ = P , .05, ∗∗ = P , .01, ∗∗∗ = P , .001. All data are means 6 standard errors of measurement.
The average clinical score at the time of imaging was similar between the groups (P = .83, Fig 4), suggesting adequate randomization. To evaluate disease activity at the relapse, we imaged the same mice several days later when they developed new symptoms. With MPOGd, we were able to detect significantly more lesions than with DTPA-Gd (9.0
6 4.0 vs 2.7 6 2.1, P = .03, Fig 4). When we compared images obtained at remission and at the first relapse, we also found that MPO-Gd helped detect more new lesions (3.8 6 2.2 vs 1.7 6 0.6, P = .047, Fig 4). Disease severity, as demonstrated by the clinical score at the first relapse (P = .69, Fig 4) and the cumulative clinical score at the first
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relapse (P = .58, Fig 4) were similar between the groups.
Inflammatory Cell Subsets in Chronic EAE At flow cytometry, we detected an increase in MPO-positive leukocytes at remission, when disease is thought to be silent, and at relapse compared with leukocytes in sham-protocol mice (P , .029 485
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Figure 4
Figure 4: MPO-Gd MR imaging in chronic EAE. Mice were imaged at remission (between the acute stage and the first relapse) (top row) and at the first relapse (bottom row) with either MPO-Gd (n = 6) or DTPA-Gd (n = 5). Arrows = lesions seen at remission; arrowheads = lesions newly seen at relapse. Results of image quantification (number of lesions and number of new lesions [remission vs relapse]) and clinical severity at remission and relapse are presented. ∗ = P , .05, ∗∗ = P , .01. All data are means 6 standard errors of the measurement.
vs remission, P = .001 vs relapse; Fig 5, A). Similar to early subacute disease, most MPO-expressing cells were neutrophils (63%), followed by Ly-6Chigh monocytes (30%). Resting microglia (3%) and lymphocytes (1%) were not a significant source of MPO (Fig 5, A). In line with these findings, we detected an increase in Ly-6Chigh inflammatory monocytes at remission and at relapse compared with those in sham-protocol mice (P = .028 for remission, P = .006 for relapse; Fig 5, B). Neutrophils also increased at remission and relapse compared with those in sham-protocol mice (P = .06 for remission, P = .01 for relapse; Fig 5, B). Finally, the number of MPOpositive cells at relapse correlated well with MPO-Gd lesion count (r = 0.93, P = .001; Fig E5, B [online]).
Discussion In this study, we showed that MPO-Gd helped detect early, subclinical disease 486
better than DTPA-Gd, findings that correlate with early migration of MPO-secreting monocytes and neutrophils into the brain. In addition, MPO-Gd also reveals subclinical disease activity at remission, when disease is thought to be silent, and also helps detect chronic disease better than DTPA-Gd, demonstrating the highly dynamic nature of this disease. Finally, we demonstrated that MPO-secreting neutrophils and Ly-6Chigh inflammatory monocytes are actively present in the brain even at remission, when disease is thought to be silent, and at the first relapse. These underlying inflammatory immune changes are detectable by using MPO-Gd MR imaging but not conventional MR imaging (DTPA enhanced or T2 weighted). Early diagnosis of active MS with prompt treatment has been found to delay clinical relapses and decrease axonal loss (22–24). However, early detection can be challenging, especially in patients after the first relapse, because
conventional DTPA-Gd–enhanced MR imaging reveals BBB breakdown, not necessarily inflammation, and leads to underestimation of the lesion burden (7). It has been demonstrated that leukocyte infiltration and BBB breakdown are two different processes and can occur independently of each other (25– 27). In addition, detection of disease activity between relapses is crucial, because the number or severity of relapses does not correlate with disease progression (28). In contrast, persistent inflammation during clinical and radiologic remission correlates with disease progression (29), and inflammation in the normal-appearing white matter contributes more strongly to disability than the T2 lesion load (30,31). An imaging test that specifically assesses inflammatory disease activity would help to circumvent these problems. The present data demonstrate that MPO-Gd is more sensitive for the detection of inflammatory lesions before symptoms become evident and at
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Figure 5
Figure 5: Inflammatory cell subsets in chronic EAE. A, MPO-expressing cells remain present and/or continue to infiltrate the brain at remission (n = 5) and relapse (n = 5). The majority of MPO-expressing cells are neutrophils (red) and inflammatory (Ly-6Chigh) monocytes (blue). Microglia and lymphocytes do not significantly contribute to MPO-expressing cells. B, Inflammatory (Ly-6Chigh) monocytes (blue) and neutrophils (red) remain elevated at remission and even more so at relapse. ∗ = P , .05, ∗∗ = P , .01. All data are means 6 standard errors of measurement.
the remission stage, and that this is because of an early influx of MPO-secreting myeloid cells to the brain. MPO-Gd– enhancing early lesions are not seen on T2-weighted images, which mostly show edema and/or demyelination (32), suggesting that MPO-expressing infiltrating
cells are detected at a very early time point of the inflammation cascade. We see two possibilities of how MPO-Gd might help detect lesions not seen with DTPA-Gd: (a) given that DTPA-Gd is not overly sensitive to subtle BBB breakdown (33,34) and activated MPO-Gd
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results in amplified signal and increased sensitivity, subtle BBB breakdown could still allow enough MPO-Gd to enter the brain to report on active inflammation and (b) inflammatory cells might release MPO during adherence and extravasation. Because MPO is well known to 487
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bind to endothelium through its positive charge (35), MPO-Gd would then identify these endothelial “footprints” left by cell migration. MPO-Gd–enhancing lesions are likely different from lesions depicted by iron oxide nanoparticles or the gadofluorine M (Gf) imaging agents previously tested in EAE. In a pilot study, Gf depicted more lesions than did DTPA-Gd in acute EAE (34). Gf enhancement exactly matched Evans blue leakage, a marker for BBB breakdown. Indeed, it was shown that Gf is mostly bound to albumin and passively diffuses through an impaired BBB, where it is retained (36). Conversely, iron oxide nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO) are thought to be phagocytosed by inflammatory cells en route to the brain. USPIO-enhanced MR imaging showed higher sensitivity for the detection of EAE than DTPA-Gd (37), and USPIO-positive lesions did not always demonstrate DTPA-Gd enhancement, and vice versa (38). In 14 patients with MS, of 188 USPIO-positive lesions, 144 did not enhance with DTPA-Gd, suggesting that phagocytic cell migration to the brain is independent of BBB breakdown (39). However, it remains unclear which cells take up USPIO in MS and whether these cells are pro or anti-inflammatory. Settles et al (40) demonstrated that both pro- and anti-inflammatory human monocytes readily phagocytose iron oxide particles of different sizes. In MS, macrophages that have phagocytosed myelin are of the anti-inflammatory (repair) M2 phenotype (41). Hence, USPIO uptake might actually represent a combination of damage and repair. This distinction becomes especially important when assessing disease activity and effects of therapy. In the present study we have shown that MPO-Gd is more sensitive than DTPAGd and helps detect MPO secreted by infiltrating proinflammatory monocytes and neutrophils. Because MPO is proinflammatory and damaging (42,43), and anti-inflammatory (Ly-6Clow) monocytes or M2 macrophages do not substantially express MPO (44,45), MPO-Gd allows for selective detection of lesions containing proinflammatory monocytes and 488
neutrophils. Therefore, it may prove to be a better imaging biomarker for assessing disease progression and/or therapeutic response than USPIO. However, future studies combining MPO-Gd with USPIO could help to identify and localize different inflammatory and reparative cell subsets. Shortcomings of the present study included the fact that MPO-Gd and DTPA-Gd could not be compared in the same animals at the same time. Nevertheless, sampling bias was avoided by randomization, and clinical disease course and severity were similar in both groups at the acute and chronic stages. The specificity of MPO-Gd has been proven by imaging MPO knockout mice, in which no specific signal could be detected (46,47). Here, MPO-Gd signal matched areas with MPO-positive cells at histologic examination, and the timing of MPO-Gd–enhancing lesions correlated with the infiltration of MPO-positive inflammatory cells. Last, although EAE is the most widely used animal model of MS, shares many pathologic features with human disease, and has led to development of several clinically used therapeutics (eg, natalizumab, glatiramer acetate) (48), it should be recognized that it is not a perfect model of human MS (49). In conclusion, MPO imaging helps detect inflammatory lesions in subclinical acute and chronic EAE, which coincides temporally and spatially with the presence of MPO-secreting proinflammatory leukocytes in the brain. MPOGd enhancing lesions are seen without overt BBB breakdown (conventional gadolinium enhancement) or edema/ demyelination (T2-weighted MR imaging). MPO-secreting leukocytes are active even in remission, when disease is thought to be silent and clinical recovery occurs. On translation, MPO-Gd might allow for detection of the proinflammatory disease burden in MS, with higher specificity and sensitivity than conventional MR imaging. Acknowledgments: We thank Cuihua Wang, PhD, for the synthesis of MPO-Gd. Disclosures of Conflicts of Interest: B.P. Activities related to the present article: has received
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a doctoral fellowship from the Ernst Schering Foundation (Berlin, Germany). Activities not related to the present article: none to disclose. Other relationships: none to disclose. L.B. disclosed no relevant relationships. G.R.W. disclosed no relevant relationships. Y.I. disclosed no relevant relationships. M.A. disclosed no relevant relationships. D.L. disclosed no relevant relationships. S.S. disclosed no relevant relationships. K.L.C.H. disclosed no relevant relationships. A.H.J. disclosed no relevant relationships. J.W.C. Activities related to the present article: none to disclose. Activities not related to the present article: none to disclose. Other relationships: has been issued U.S. Patent 8,153,784 for the imaging agent used in this study.
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18. Chen JW, Querol Sans M, Bogdanov A Jr, Weissleder R. Imaging of myeloperoxidase in mice by using novel amplifiable paramagnetic substrates. Radiology 2006;240(2):473–481. 19. Chen JW, Breckwoldt MO, Aikawa E, Chiang G, Weissleder R. Myeloperoxidase-targeted imaging of active inflammatory lesions in murine experimental autoimmune encephalomyelitis. Brain 2008;131(Pt 4):1123–1133. 20. Forghani R, Wojtkiewicz GR, Zhang Y, et al. Demyelinating diseases: myeloperoxidase as an imaging biomarker and therapeutic target. Radiology 2012;263(2):451–460. 21. Pino PA, Cardona AE. Isolation of brain and spinal cord mononuclear cells using percoll gradients. J Vis Exp 2011 (48). 22. Jacobs LD, Beck RW, Simon JH, et al. Intramuscular interferon beta-1a therapy initiated during a first demyelinating event in multiple sclerosis. CHAMPS Study Group. N Engl J Med 2000;343(13):898–904. 23. Comi G, Filippi M, Barkhof F, et al. Effect of early interferon treatment on conversion to definite multiple sclerosis: a randomised study. Lancet 2001;357(9268):1576–1582. 24. Comi G, Martinelli V, Rodegher M, et al. Effect of glatiramer acetate on conversion to clinically definite multiple sclerosis in patients with clinically isolated syndrome (PreCISe study): a randomised, double-blind, placebo-controlled trial. Lancet 2009;374(9700):1503–1511. 25. Perry VH, Anthony DC, Bolton SJ, Brown HC. The blood-brain barrier and the in-
31. Traboulsee A, Dehmeshki J, Peters KR, et al. Disability in multiple sclerosis is related to normal appearing brain tissue MTR histogram abnormalities. Mult Scler 2003;9(6):566–573. 32. Filippi M, Rocca MA, Barkhof F, et al. Association between pathological and MRI findings in multiple sclerosis. Lancet Neurol 2012;11(4):349–360. 33. Silver NC, Good CD, Barker GJ, et al. Sensitivity of contrast enhanced MRI in multiple sclerosis: effects of gadolinium dose, magnetization transfer contrast and delayed imaging. Brain 1997;120(Pt 7):1149–1161. 34. Bendszus M, Ladewig G, Jestaedt L, et al. Gadofluorine M enhancement allows more sensitive detection of inflammatory CNS lesions than T2-w imaging: a quantitative MRI study. Brain 2008;131(Pt 9):2341–2352. 35. Klinke A, Nussbaum C, Kubala L, et al. Myeloperoxidase attracts neutrophils by physical forces. Blood 2011;117(4):1350–1358. 36. Meding J, Urich M, Licha K, et al. Magnetic resonance imaging of atherosclerosis by targeting extracellular matrix deposition with Gadofluorine M. Contrast Media Mol Imaging 2007;2(3):120–129. 37. Dousset V, Ballarino L, Delalande C, et al. Comparison of ultrasmall particles of iron oxide (USPIO)-enhanced T2-weighted, conventional T2-weighted, and gadoliniumenhanced T1-weighted MR images in rats with experimental autoimmune encepha-
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40. Settles M, Etzrodt M, Kosanke K, et al. Different capacity of monocyte subsets to phagocytose iron-oxide nanoparticles. PLoS ONE 2011;6(10):e25197. 41. Boven LA, Van Meurs M, Van Zwam M, et al. Myelin-laden macrophages are anti-inflammatory, consistent with foam cells in multiple sclerosis. Brain 2006;129(Pt 2): 517–526. 42. Davies MJ. Myeloperoxidase-derived oxidation: mechanisms of biological damage and its prevention. J Clin Biochem Nutr 2011; 48(1):8–19. 43. Nauseef WM. Contributions of myeloper oxidase to proinflammatory events: more than an antimicrobial system. Int J Hematol 2001;74(2):125–133. 44. Swirski FK, Nahrendorf M, Etzrodt M, et al. Identification of splenic reservoir monocytes and their deployment to inflammatory sites. Science 2009;325(5940):612–616. 45. Sugiyama S, Okada Y, Sukhova GK, Vir mani R, Heinecke JW, Libby P. Macrophage myeloperoxidase regulation by granulocyte macrophage colony-stimulating factor in human atherosclerosis and implications in acute coronary syndromes. Am J Pathol 2001;158(3):879–891. 46. Nahrendorf M, Sosnovik D, Chen JW, et al. Activatable magnetic resonance imaging agent reports myeloperoxidase activity in healing infarcts and noninvasively detects the antiinflammatory effects of atorvastatin on ischemia-reperfusion injury. Circulation 2008;117(9):1153–1160. 47. Breckwoldt MO, Chen JW, Stangenberg L, et al. Tracking the inflammatory response in stroke in vivo by sensing the enzyme myeloperoxidase. Proc Natl Acad Sci U S A 2008; 105(47):18584–18589. 48. Steinman L, Zamvil SS. How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis. Ann Neurol 2006;60(1):12–21. 49. Sriram S, Steiner I. Experimental allergic encephalomyelitis: a misleading model of multiple sclerosis. Ann Neurol 2005;58(6): 939–945.
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Multiple Sclerosis: Myeloperoxidase Immunoradiology Improves Detection of Acute and Chronic Disease in Experimental Model1 Benjamin Pulli, MD Lionel Bure, MD Gregory R. Wojtkiewicz, MSc Yoshiko Iwamoto, BSc Muhammad Ali, MD Dan Li, MD Stefan Schob, MD Kevin Li-Chun Hsieh, MD2 Andreas H. Jacobs, MD John W. Chen, MD, PhD
Purpose:
To test if MPO-Gd, a gadolinium-based magnetic resonance (MR) imaging probe that is sensitive and specific for the proinflammatory and oxidative enzyme myeloperoxidase (MPO), which is secreted by certain inflammatory cells, is more sensitive than diethylenetriaminepentaacetic acid (DTPA)-Gd in revealing early subclinical and chronic disease activity in the brain in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis.
Materials and Methods:
The protocol for animal experiments was approved by the institutional animal care committee. A total of 61 female SJL mice were induced with EAE. Mice underwent MPOGd– or DTPA-Gd–enhanced MR imaging on days 6, 8, and 10 after induction, before clinical disease develops, and during chronic disease at remission and the first relapse. Brains were harvested at these time points for flow cytometric evaluation of immune cell subtypes and immunohistochemistry. Statistical analysis was performed, and P , .05 was considered to indicate a significant difference.
Results:
MPO-Gd helps detect earlier (5.2 vs 2.3 days before symptom onset, P = .004) and more (3.1 vs 0.3, P = .008) subclinical inflammatory lesions compared with DTPAGd, including in cases in which there was no evidence of overt blood-brain barrier (BBB) breakdown detected with DTPA-Gd enhancement. The number of MPO-Gd–enhancing lesions correlated with early infiltration of MPOsecreting monocytes and neutrophils into the brain (r = 0.91). MPO-Gd also helped detect more lesions during subclinical disease at remission (5.5 vs 1.3, P = .006) and at the first relapse (9.0 vs 2.7, P = .03) than DTPA-Gd, which also correlated well with the presence and accumulation of MPO-secreting inflammatory cells in the brain (r = 0.93).
Conclusion:
MPO-Gd specifically reveals lesions with inflammatory monocytes and neutrophils, which actively secrete MPO. These results demonstrate the feasibility of detection of subclinical inflammatory disease activity in vivo, which is different from overt BBB breakdown.
1
From the Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, 185 Cambridge St, Boston, MA 02114 (B.P., L.B., G.R.W., Y.I., M.A., D.L., S.S., K.L.C.H., J.W.C.); Department of Radiology, Massachusetts General Hospital, Boston, Mass (B.P., J.W.C.); and European Institute for Molecular Imaging, University of Münster, Münster, Germany (A.H.J.). From the 2012 RSNA Annual Meeting. Received June 25, 2014; revision requested August 6; revision received August 11; accepted August 27; final version accepted September 19. B.P. supported by a fellowship from the Ernst Schering Foundation (Berlin, Germany) and a grant from the National Natural Science Foundation (Beijing, China). Address correspondence to J.W.C. (e-mail: [email protected]).
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q
Current address: Medical Imaging Center, Taipei Medical University Hospital, Taipei, Taiwan, Republic of China. 2
Online supplemental material is available for this article.
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M
ultiple sclerosis (MS) is the most common cause of disability in young adults (1). Although the pathogenesis of MS remains elusive, the presence of CD4+ autoreactive T-lymphocytes in the blood and brain of patients with MS suggest that it is a T-lymphocyte driven disease (2). Demyelination and axonal damage, however, are more likely caused by innate immune cells such as monocytes, macrophages, and neutrophils (2). Accordingly, in experimental autoimmune encephalomyelitis (EAE), the most commonly used experimental model of MS, depletion or inactivation of monocytes (3,4), microglia (5), and neutrophils (6) effectively suppresses disease. Early diagnosis of active MS with prompt treatment has been found to delay clinical relapses and decrease axonal loss. Magnetic resonance (MR) imaging is routinely used to assess disease
Advances in Knowledge nn Molecular MR imaging of myeloperoxidase (MPO) helps detect inflammatory lesions in experimental autoimmune encephalomyelitis (EAE) earlier (5.2 vs 2.3 days before symptom onset, P = .004), when there is no overt evidence of blood-brain barrier (BBB) breakdown (as detected by means of conventional gadolinium enhancement) or edema and/or demyelination (as detected with T2-weighted imaging).
activity in patients with MS but underreports inflammatory lesions, especially in the cortex (7). Moreover, only weak correlation has been found between MR imaging lesion volume and disease progression to disability (8,9). Gadoliniumenhancing lesions reflect blood-brain barrier (BBB) breakdown rather than active inflammation and do not predict relapse rate (10). This clinical-radiologic paradox might be explained by inflammatory activity behind an intact or partially repaired BBB (11,12). Myeloperoxidase (MPO) is an important oxidative enzyme secreted by innate immune cells (13). A higher-expressing MPO phenotype has been associated with early-onset MS (14), and MPO expression has been detected in white (15) and gray (16) matter plaques in patients with MS. The MPO-targeted MR imaging probe MPO-Gd (17,18) has been used to demonstrate MPO activity in vivo in EAE (19) and has been shown to be sensitive to treatment with a preclinical MPO inhibitor (20). However, it remains unclear if MPO-Gd can help detect acute and chronic subclinical inflammation, when the BBB is mostly closed, with higher sensitivity than diethylenetriaminepentaacetic acid (DTPA)-Gd. The aim of this study was to test if MPO-Gd is more sensitive than DTPAGd in the detection of early subclinical and chronic disease activity in the brain in EAE, a mouse model of MS.
nn MPO imaging also helps detect more lesions in disease remission (5.5 vs 1.3, P = .006), when disease is thought to be silent, and at the first relapse (9.0 vs 2.7, P = .03). nn MPO imaging results correlate well (r = 0.91 for subclinical acute and r = 0.93 for chronic EAE) with the presence of MPOsecreting proinflammatory monocytes and neutrophils in the brain, which migrate into the brain before the onset of clinical symptoms and remain active in disease remission. Radiology: Volume 275: Number 2—May 2015 n radiology.rsna.org
Implications for Patient Care nn On translation, MPO imaging could allow specific detection of the proinflammatory disease burden in multiple sclerosis (MS), extending on information that conventional T2-weighted imaging (edema/demyelination), gadolinium (BBB breakdown), and iron oxide (infiltration of phagocytic cells that can cause damage or repair) provide. nn Because persistent inflammation during clinical and radiologic remission correlates with disease progression in MS, MPO imaging may help predict progression to disability.
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Materials and Methods EAE Induction The protocol for animal experiments was approved by the institutional animal care committee. EAE was induced in 61 female SJL mice that were 6–10 weeks old (B.P., with 4 years of experience in EAE induction) with synthetic proteolipid protein (PLP139–151; NeoBioscience, Cambridge, Mass), as previously described (19,20). Briefly, each mouse was injected subcutaneously with a suspension containing 400 mg Mycobacterium tuberculosis H37RA (Difco) and 100 mg PLP139–151 in one part complete Freund adjuvant (CFA; Sigma, St Louis, Mo) mixed with one part phosphate-buffered saline. Within 2 hours of induction and on day 2, mice received 0.1 mg pertussis toxin (Sigma) intravenously. Sham-protocol mice were induced with identical steps, except that PLP was not used. With this induction protocol, disease onset was typically around days 11–13,
Published online before print 10.1148/radiol.14141495 Content codes: Radiology 2015; 275:480–489 Abbreviations: BBB = blood-brain barrier DTPA = diethylenetriaminepentaacetic acid EAE = experimental autoimmune encephalomyelitis MPO = myeloperoxidase MS = multiple sclerosis USPIO = ultrasmall superparamagnetic iron oxide Author contributions: Guarantors of integrity of entire study, B.P., G.R.W., S.S., J.W.C.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; agrees to ensure any questions related to the work are appropriately resolved, all authors; literature research, B.P., L.B., G.R.W., S.S., K.L.C.H., J.W.C.; experimental studies, B.P., L.B., G.R.W., Y.I., M.A., D.L., S.S., K.L.C.H., J.W.C.; statistical analysis, B.P., L.B., G.R.W.; and manuscript editing, B.P., G.R.W., M.A., S.S., K.L.C.H., A.H.J., J.W.C. Funding: This research was supported by the National Institutes of Health (grants R01-NS070835 and R01-NS072167). Conflicts of interest are listed at the end of this article. See also Science to Practice in this issue.
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with a first relapse occurring around days 24–30 after induction. The beginning of our study was induction with EAE, and mice were sacrificed either on days 6, 8, or 10 after induction (early subclinical disease), at remission (days 20–25 after induction), or at the first relapse (days 24–30 after induction.
Clinical Scoring Mice were checked daily for any signs of EAE. Weights were recorded daily. Clinical assessment for disease activity was performed by using the standard five-point staging system for EAE with the following scale (B.P. and D.L., who were blinded to which imaging agent was used): score 0, no signs; score 1, complete tail limpness without limb weakness; score 2, partial limb weakness and impaired righting reflex; score 3, at least one limb with complete paralysis; score 4, complete paralysis; and score 5, moribund. MPO-Gd Molecular MR Imaging To measure the specific MPO activity within the inflamed brain in vivo in living animals, we performed serial MPO-Gd (bis-5-hydroxytryptamide-diethylenetriaminepentaacetate gadolinium) molecular MR imaging in EAE mice (19,20). Early, subclinical lesions were detected on day 6, 8 and 10, when mice were asymptomatic. Lesions in remission and those in the first relapse were detected between days 18–23 and days 24–30, respectively, depending on the clinical score. Mice were considered to be in remission when the clinical score improved more than 1 point to either 0 or 1. A relapse was defined as weight loss of at least 0.5 g and an increase in the clinical score of at least 1 (Fig E1, A [online]). MPO-Gd is an activatable MR imaging agent that reports extracellular MPO activity in vivo with high specificity and sensitivity (17,18). MPO-Gd was synthesized as previously described (17–19). MR imaging was performed by using an animal 4.7-T MR imaging unit (Bruker, Billerica, Mass) consisting of mouse coronal T2-weighted images and T1-weighted images obtained before and after intravenous administration of MPO-Gd (0.3 mmol per kilogram of 482
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body weight) or DTPA-Gd (0.3 mmol/ kg). Mice were randomized to undergo imaging with either MPO-Gd or DTPAGd, as seen in the imaging flowchart (Fig E1b [online]). MR images were independently evaluated by two authors (B.P. and L.B., with 4 and 3 years of experience in interpreting MR images, respectively), who were blinded to the imaging agent used, the clinical scores of the mice, and the sham versus experimental group. Total lesion volume, total lesion count, and mean lesion volume were calculated.
cell numbers of different cell populations were calculated as total cells multiplied by the percentage within the respective cell population gate. Data were acquired with a flow cytometer (LSR II; BD Biosciences) and were analyzed with dedicated software (FlowJo 887; Tree Star, Ashland, Ore). Flow cytometric analysis was performed by one author (B.P., with 4 years of experience in flow cytometry of brain leukocytes). To evaluate the inflammatory cell infiltration specific to EAE, mice with EAE were compared with sham-protocol mice.
Isolation of Brain Inflammatory Cells and Flow Cytometry Mice were transcardially perfused with 20 mL ice-cold phosphate-buffered saline. Brain leukocytes were extracted by density centrifugation over a discontinuous Percoll gradient, as previously described (21). Briefly, brain tissue was mechanically ground in a glass tissue homogenizer in 8 mL of 30% Percoll solution (GE Healthcare, Boston, Mass), filtered through a 40-mm cell strainer (BD Biosciences, San Jose, Calif), and overlaid over 3 mL 70% Percoll. The density gradient was centrifuged at 650 g for 25 minutes at 18°C. After centrifugation, a thick myelin-containing layer at the top was removed, and the brain leukocytes at the 30/70 interface were collected. The cells were then counted and stained for flow cytometry. All antibodies were purchased from BD Bioscience unless otherwise indicated. The following antibodies were used: anti-CD90, 53–2.1; anti-NK1.1, PK136; anti-B220, RA3–6B2; anti-CD49b, DX5; anti-Ly-6G, IA8; anti-TER-119, TER-119; anti-CD11b, M1/70; anti-Ly-6C, AL-21; and anti-F4/80, C1:A3–1 (BioLegend, San Diego, Calif). For intracellular staining of MPO, cells were fixed and permeabilized (Cytofix/Cytoperm; BD Bioscience) after staining for cell surface markers; this was followed by staining for MPO using anti-MPO (clone 8F4; Hycult Biotech, Plymouth Meeting, Pa). When appropriate, a streptavidin-conjugated secondary antibody (eBioscience, San Diego, Calif) was used to label biotinylated antibodies. Our gating strategy can be seen in Figure E2 (online). The
Histopathologic Analysis Histopathologic analysis was performed to assess for MPO-expressing inflammatory cells. Fresh-frozen 5-mm brain sections were incubated in 1% hydrogen peroxide solution for 10 minutes and were blocked with 4% normal rabbit or goat serum for 30 minutes at room temperature. The sections were incubated with anti-MPO (Ab-1; Thermo Fisher Scientific, Waltham, Mass). A biotinylated secondary antibody (Vector Laboratories, Burlingame, Mass) was used. Statistical Analysis Results are reported as means 6 standard deviations. The data were tested for normality by using the D’AgostinoPearson normality test. If normality was not rejected, the t test was used. Otherwise, we used the nonparametric Mann-Whitney U test. The Fisher exact test was used to compare the number of mice found to have positive MR imaging findings with MPO-Gd versus DTPA-Gd. The Pearson correlation coefficient was calculated for MPO-Gd lesion counts versus number of MPOexpressing cells at flow cytometry. P , .05 was considered to indicate a significant difference. We used software for statistical analysis (GraphPad Prism 5; GraphPad Software). Results Detection of Early, Subclinical Disease with MPO-Gd MR Imaging MPO-Gd–enhanced molecular MR imaging helped detect at least one
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inflammatory lesion on day 8.0 6 1.1, while DTPA-Gd enhanced MR imaging was first positive on day 9.7 6 0.8 (P = .008). MPO-Gd first showed lesions 5.3 days 6 1.3 versus 2.3 days 6 1.0 before the onset of clinical symptoms (P = .004, Fig 1). On day 8, MPO-Gd showed significantly more lesions than DTPA-Gd (3.1 6 2.0 vs 0.3 6 0.8, P = .008). We also found that the average lesion size at the time of first detection was much smaller with MPO-Gd (4.6 mm3 6 3.1)
than with DTPA-Gd (38.5 6 31 mm3, P = .009, Fig 1). With MPO-Gd, 13% (one of eight) of mice were found to have a positive MR imaging result on day 6; by day 8, this number increased to 88% (seven of eight). With DTPA-Gd, no lesions were found on day 6, and only one (17%) of six mice was found to have a lesion on day 8 (P = .026 for day 8 comparison between MPO-Gd and DTPAGd). By day 10, lesions were found in all mice, regardless of imaging agent (Fig
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1). To evaluate if MPO-Gd enhancement also preceded T2 signal changes, which can be suggestive of edema and/or demyelination, we compared images from mice imaged with MPO-Gd on day 8. All lesions detected on day 8 with MPO-Gd were not seen on T2-weighted MR images, and no other lesions were detected (3.1 6 2.0 vs 0.0 6 0.0 lesions for MPOGd vs T2, P = .002, Fig 1). Representative images from mice imaged on day 8 with MPO-Gd and
Figure 1
Figure 1: Earliest detection of inflammation with MPO-Gd MR imaging in subclinical EAE. Molecular MR imaging with MPO-Gd (upper row) and DTPA-Gd (lower row) on day 6 (left) and day 8 (right) after disease induction reveals a small inflammatory lesion visible with MPO-Gd but not with DTPA-Gd (arrow). The inflammatory lesion seen with MPO-Gd was determined to correspond to an area of MPO-positive cells at immunohistochemistry, while no other areas containing MPO-positive cells were seen. MR image quantification revealed that MPO-Gd helps detect lesions earlier (more days before symptom onset, and with a higher percentage of positive MR imaging findings on postinduction day 8) and helps detect more and smaller lesions than DTPA-Gd. Day of disease onset was similar in the two groups. MPO-Gd also revealed more subclinical lesions than T2-weighted imaging (n = 8 for MPO-Gd and T2-weighted imaging, n = 6 for DTPA-Gd). ∗ = P , .05, ∗∗ = P , .01. p.i. = Postinduction. All data are means 6 standard errors of measurement. Radiology: Volume 275: Number 2—May 2015 n radiology.rsna.org
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Figure 2
Figure 2: Subclinical, early inflammatory lesions detected with MPO-Gd MR imaging on day 8 after EAE induction. A, Images show inflammatory lesions (arrows) in mice imaged with MPO-Gd (top row) and the absence of overt BBB breakdown as depicted by DTPA-Gd (bottom row). B, Inflammatory lesions (arrows) in mice imaged with MPO-Gd are detected on postcontrast T1-weighted (top) but not precontrast T2-weighted images (bottom).
DTPA-Gd can be seen in Figure 2, A. Postcontrast images and T2-weighted images from mice imaged on day 8 are shown in Figure 2, B. To confirm the specificity of MPO-Gd, we performed immunohistochemistry for MPO, where we could confirm a lesion with MPOpositive cells at a location corresponding to the MR imaging results (Fig 1). In line with a successful randomization, we did not find any difference in the clinical disease severity, as shown by similar clinical score profiles (Fig E3, A [online]), disease onset (P = .50, Fig 1), and cumulative disease scores (P = .55, Fig E3, B [online]).
Inflammatory Cell Subsets in Subacute, Early EAE In line with MPO-Gd imaging findings that in 88% of mice at least one discernible lesion was detectable as early as day 8, we found a significant increase in MPO-positive leukocytes with flow cytometry on day 8 compared with those in sham-protocol mice (P = .035). Similar results were observed on day 10 484
(P = .001, Fig 3, A). However, there was no significant increase in MPO-expressing leukocytes on day 6 (P = .16), consistent with imaging results. Next, we identified the leukocyte subtypes expressing MPO. Fifty-five percent were neutrophils and 38% were Ly-6Chigh monocytes, while neither microglia (3%) nor lymphocytes (1%) contributed significantly (Fig 3, A). In line with these findings, we detected a significant increase in Ly-6Chigh monocytes on days 8 and 10 compared with those in shamprotocol mice (P = .002 for day 8 and P , .001 for day 10; Fig 3, B). A similar trend was found for neutrophils, which were also significantly increased on day 8 and 10 compared with those in shamprotocol mice (P , .029 for day 8 and P , .001 for day 10; Fig 3, B). Last, we measured the MPO content of these different cell subsets. Neutrophils were found to have the most MPO (median fluorescent intensity [MFI], 3785; P = .002), followed by Ly-6Chigh monocytes (MFI, 2069; P = .002). Microglia (MFI, 771; P = .28) and lymphocytes (MFI,
314; P = .87) were not found to express MPO (isotype control MFI, 317; Fig E4, A [online]). In the blood, neutrophils (MFI, 4252; P = .023) and Ly-6Chigh monocytes (MFI, 3303; P = .023) were found to express MPO, while Ly-6Clow monocytes did not (MFI, 1627; P = .12) compared with isotype control (MFI, 1063; Fig E4, B [online]). Finally, the number of MPO-positive cells on days 6, 8, and 10 correlated well with MPO-Gd MR imaging lesion count (r = 0.91, P = .001; Fig E5, A [online]). Cumulatively, these results form a biologic basis for our imaging findings: MPO-secreting neutrophils and Ly-6Chigh inflammatory monocytes infiltrate as early as day 8 after induction, coinciding with the time of earliest detection of inflammation at MPO-Gd MR imaging.
Detection of Chronic Disease Activity with MPO-Gd MR Imaging At remission, we found a significantly higher number of lesions with MPO-Gd than with DTPA-Gd (5.5 6 0.8 vs 1.3 6 1.0 lesions per mouse, P = .006, Fig 4).
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Figure 3
Figure 3: Inflammatory cell subsets in subacute, early EAE. A, MPO-expressing cells accumulate in the brain on days 8 and 10 after induction. About 55% of MPO-expressing cells are neutrophils (red), and 45% are inflammatory (Ly-6Chigh) monocytes (blue). Microglia (black) and lymphocytes do not significantly contribute to MPO-expressing cells (n = 6 per group). B, Inflammatory (Ly-6Chigh) monocytes (blue) and neutrophils (red) infiltrate the brain on days 8 and 10 after induction (n = 6 per group). ∗ = P , .05, ∗∗ = P , .01, ∗∗∗ = P , .001. All data are means 6 standard errors of measurement.
The average clinical score at the time of imaging was similar between the groups (P = .83, Fig 4), suggesting adequate randomization. To evaluate disease activity at the relapse, we imaged the same mice several days later when they developed new symptoms. With MPOGd, we were able to detect significantly more lesions than with DTPA-Gd (9.0
6 4.0 vs 2.7 6 2.1, P = .03, Fig 4). When we compared images obtained at remission and at the first relapse, we also found that MPO-Gd helped detect more new lesions (3.8 6 2.2 vs 1.7 6 0.6, P = .047, Fig 4). Disease severity, as demonstrated by the clinical score at the first relapse (P = .69, Fig 4) and the cumulative clinical score at the first
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relapse (P = .58, Fig 4) were similar between the groups.
Inflammatory Cell Subsets in Chronic EAE At flow cytometry, we detected an increase in MPO-positive leukocytes at remission, when disease is thought to be silent, and at relapse compared with leukocytes in sham-protocol mice (P , .029 485
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Figure 4
Figure 4: MPO-Gd MR imaging in chronic EAE. Mice were imaged at remission (between the acute stage and the first relapse) (top row) and at the first relapse (bottom row) with either MPO-Gd (n = 6) or DTPA-Gd (n = 5). Arrows = lesions seen at remission; arrowheads = lesions newly seen at relapse. Results of image quantification (number of lesions and number of new lesions [remission vs relapse]) and clinical severity at remission and relapse are presented. ∗ = P , .05, ∗∗ = P , .01. All data are means 6 standard errors of the measurement.
vs remission, P = .001 vs relapse; Fig 5, A). Similar to early subacute disease, most MPO-expressing cells were neutrophils (63%), followed by Ly-6Chigh monocytes (30%). Resting microglia (3%) and lymphocytes (1%) were not a significant source of MPO (Fig 5, A). In line with these findings, we detected an increase in Ly-6Chigh inflammatory monocytes at remission and at relapse compared with those in sham-protocol mice (P = .028 for remission, P = .006 for relapse; Fig 5, B). Neutrophils also increased at remission and relapse compared with those in sham-protocol mice (P = .06 for remission, P = .01 for relapse; Fig 5, B). Finally, the number of MPOpositive cells at relapse correlated well with MPO-Gd lesion count (r = 0.93, P = .001; Fig E5, B [online]).
Discussion In this study, we showed that MPO-Gd helped detect early, subclinical disease 486
better than DTPA-Gd, findings that correlate with early migration of MPO-secreting monocytes and neutrophils into the brain. In addition, MPO-Gd also reveals subclinical disease activity at remission, when disease is thought to be silent, and also helps detect chronic disease better than DTPA-Gd, demonstrating the highly dynamic nature of this disease. Finally, we demonstrated that MPO-secreting neutrophils and Ly-6Chigh inflammatory monocytes are actively present in the brain even at remission, when disease is thought to be silent, and at the first relapse. These underlying inflammatory immune changes are detectable by using MPO-Gd MR imaging but not conventional MR imaging (DTPA enhanced or T2 weighted). Early diagnosis of active MS with prompt treatment has been found to delay clinical relapses and decrease axonal loss (22–24). However, early detection can be challenging, especially in patients after the first relapse, because
conventional DTPA-Gd–enhanced MR imaging reveals BBB breakdown, not necessarily inflammation, and leads to underestimation of the lesion burden (7). It has been demonstrated that leukocyte infiltration and BBB breakdown are two different processes and can occur independently of each other (25– 27). In addition, detection of disease activity between relapses is crucial, because the number or severity of relapses does not correlate with disease progression (28). In contrast, persistent inflammation during clinical and radiologic remission correlates with disease progression (29), and inflammation in the normal-appearing white matter contributes more strongly to disability than the T2 lesion load (30,31). An imaging test that specifically assesses inflammatory disease activity would help to circumvent these problems. The present data demonstrate that MPO-Gd is more sensitive for the detection of inflammatory lesions before symptoms become evident and at
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Figure 5
Figure 5: Inflammatory cell subsets in chronic EAE. A, MPO-expressing cells remain present and/or continue to infiltrate the brain at remission (n = 5) and relapse (n = 5). The majority of MPO-expressing cells are neutrophils (red) and inflammatory (Ly-6Chigh) monocytes (blue). Microglia and lymphocytes do not significantly contribute to MPO-expressing cells. B, Inflammatory (Ly-6Chigh) monocytes (blue) and neutrophils (red) remain elevated at remission and even more so at relapse. ∗ = P , .05, ∗∗ = P , .01. All data are means 6 standard errors of measurement.
the remission stage, and that this is because of an early influx of MPO-secreting myeloid cells to the brain. MPO-Gd– enhancing early lesions are not seen on T2-weighted images, which mostly show edema and/or demyelination (32), suggesting that MPO-expressing infiltrating
cells are detected at a very early time point of the inflammation cascade. We see two possibilities of how MPO-Gd might help detect lesions not seen with DTPA-Gd: (a) given that DTPA-Gd is not overly sensitive to subtle BBB breakdown (33,34) and activated MPO-Gd
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results in amplified signal and increased sensitivity, subtle BBB breakdown could still allow enough MPO-Gd to enter the brain to report on active inflammation and (b) inflammatory cells might release MPO during adherence and extravasation. Because MPO is well known to 487
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bind to endothelium through its positive charge (35), MPO-Gd would then identify these endothelial “footprints” left by cell migration. MPO-Gd–enhancing lesions are likely different from lesions depicted by iron oxide nanoparticles or the gadofluorine M (Gf) imaging agents previously tested in EAE. In a pilot study, Gf depicted more lesions than did DTPA-Gd in acute EAE (34). Gf enhancement exactly matched Evans blue leakage, a marker for BBB breakdown. Indeed, it was shown that Gf is mostly bound to albumin and passively diffuses through an impaired BBB, where it is retained (36). Conversely, iron oxide nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO) are thought to be phagocytosed by inflammatory cells en route to the brain. USPIO-enhanced MR imaging showed higher sensitivity for the detection of EAE than DTPA-Gd (37), and USPIO-positive lesions did not always demonstrate DTPA-Gd enhancement, and vice versa (38). In 14 patients with MS, of 188 USPIO-positive lesions, 144 did not enhance with DTPA-Gd, suggesting that phagocytic cell migration to the brain is independent of BBB breakdown (39). However, it remains unclear which cells take up USPIO in MS and whether these cells are pro or anti-inflammatory. Settles et al (40) demonstrated that both pro- and anti-inflammatory human monocytes readily phagocytose iron oxide particles of different sizes. In MS, macrophages that have phagocytosed myelin are of the anti-inflammatory (repair) M2 phenotype (41). Hence, USPIO uptake might actually represent a combination of damage and repair. This distinction becomes especially important when assessing disease activity and effects of therapy. In the present study we have shown that MPO-Gd is more sensitive than DTPAGd and helps detect MPO secreted by infiltrating proinflammatory monocytes and neutrophils. Because MPO is proinflammatory and damaging (42,43), and anti-inflammatory (Ly-6Clow) monocytes or M2 macrophages do not substantially express MPO (44,45), MPO-Gd allows for selective detection of lesions containing proinflammatory monocytes and 488
neutrophils. Therefore, it may prove to be a better imaging biomarker for assessing disease progression and/or therapeutic response than USPIO. However, future studies combining MPO-Gd with USPIO could help to identify and localize different inflammatory and reparative cell subsets. Shortcomings of the present study included the fact that MPO-Gd and DTPA-Gd could not be compared in the same animals at the same time. Nevertheless, sampling bias was avoided by randomization, and clinical disease course and severity were similar in both groups at the acute and chronic stages. The specificity of MPO-Gd has been proven by imaging MPO knockout mice, in which no specific signal could be detected (46,47). Here, MPO-Gd signal matched areas with MPO-positive cells at histologic examination, and the timing of MPO-Gd–enhancing lesions correlated with the infiltration of MPO-positive inflammatory cells. Last, although EAE is the most widely used animal model of MS, shares many pathologic features with human disease, and has led to development of several clinically used therapeutics (eg, natalizumab, glatiramer acetate) (48), it should be recognized that it is not a perfect model of human MS (49). In conclusion, MPO imaging helps detect inflammatory lesions in subclinical acute and chronic EAE, which coincides temporally and spatially with the presence of MPO-secreting proinflammatory leukocytes in the brain. MPOGd enhancing lesions are seen without overt BBB breakdown (conventional gadolinium enhancement) or edema/ demyelination (T2-weighted MR imaging). MPO-secreting leukocytes are active even in remission, when disease is thought to be silent and clinical recovery occurs. On translation, MPO-Gd might allow for detection of the proinflammatory disease burden in MS, with higher specificity and sensitivity than conventional MR imaging. Acknowledgments: We thank Cuihua Wang, PhD, for the synthesis of MPO-Gd. Disclosures of Conflicts of Interest: B.P. Activities related to the present article: has received
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a doctoral fellowship from the Ernst Schering Foundation (Berlin, Germany). Activities not related to the present article: none to disclose. Other relationships: none to disclose. L.B. disclosed no relevant relationships. G.R.W. disclosed no relevant relationships. Y.I. disclosed no relevant relationships. M.A. disclosed no relevant relationships. D.L. disclosed no relevant relationships. S.S. disclosed no relevant relationships. K.L.C.H. disclosed no relevant relationships. A.H.J. disclosed no relevant relationships. J.W.C. Activities related to the present article: none to disclose. Activities not related to the present article: none to disclose. Other relationships: has been issued U.S. Patent 8,153,784 for the imaging agent used in this study.
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