Myeloperoxidase-rich Ly-6C+ myeloid cells infiltrate allografts and contribute to an imaging signature of organ rejection in mice

Abstract

Rates of graft rejection are high among recipients of heart transplants. The onset and progression of clinically significant heart transplant rejection are currently monitored by serial biopsy, but this approach is highly invasive and lacks sensitivity. Here, we have developed what we believe to be a new technique to measure organ rejection noninvasively that involves the exploration of tissue-infiltrating leukocytes as biomarker sources for diagnostic imaging. Specifically, we profiled the myeloid response in a murine model of heart transplantation with the aim of defining and validating an imaging signature of graft rejection. Ly-6Chi monocytes, which promote inflammation, accumulated progressively in allografts but only transiently in isografts. Ly-6Clo monocytes, which help resolve inflammation, did not accumulate, although they composed the majority of the few remaining monocytes in isografts. The persistence of Ly-6Chi monocytes in allografts prompted us to screen for a Ly-6Chi monocyte-associated imaging marker. Low-density array data revealed that Ly-6Chi monocytes express 10-fold higher levels of myeloperoxidase (MPO) than Ly-6Clo monocytes. Noninvasive magnetic resonance imaging of MPO with an MPO-activatable Gd-chelate revealed a spatially defined T1-weighted signal in rejected allografts but not in isografts or MPO-deficient allograft recipients. Flow cytometry, enzymography, and histology validated the approach by mapping MPO activity to Ly-6Chi monocytes and neutrophils. Thus, MPO imaging represents a potential alternative to the current invasive clinical standard by which transplants are monitored.

Figure 1. Myeloid cell profile in transplant rejection.

(A) Flow cytometric analysis of cell suspensions retrieved from native hearts and iso- and allografted hearts 7 days after transplantation. Lin, CD90/B220/CD49b/NK1.1/Ly-6G. Histograms on right depict the level of Ly-6C expression in respective populations. A representative experiment of 5 is shown. Tx, transplantation. (B) Time course of myeloid cell presence in heart and blood of allograft (black circles) and isograft (white circles) recipients. Animals receiving no transplants served as controls. (C) Ratio of Ly-6C^hi vs. Ly-6C^lo monocytes in the native heart, in isografts and allografts, and in blood. Data are shown as mean ± SEM; n = 3–5 per group.

Figure 2. MPO levels in leukocytes retrieved from blood and transplants.

(A) Flow cytometric intracellular MPO staining on lymphocytes, neutrophils, Ly-6C^hi monocytes, and Ly-6C^lo monocytes. 1 of 3 representative experiments is shown. Numbers indicate percentage of cells in respective gates. (B) Relative contribution of leukocytes to overall MPO expression in allografts and isografts. The area of the pie chart reflects overall MPO content per mg tissue, which is the product of per cell MPO content and the abundance of each cell type in the heart, as determined by flow cytometry. (C) MPO activity (n = 3) of lysed sorted leukocytes.

Figure 3. MRI of MPO activity.

(A and B) Orientation of the heterotopic graft and localizer MRI. (C) Change of CNR from before to 2 hours after MPO-Gd injection in wild-type and MPO-deficient mice 7 days after allograft transplantation. Data are shown as mean ± SEM; n = 4–9 per group; *P = 0.02. (D and E) Representative short axis views of allografts 2 hours after injection of MPO-Gd.

Figure 4. Kinetics and distribution of enhancement.

(A and B) T1-weighted short axis MRI of allo- and isografts 7 days after transplantation before and up to 120 minutes after injection of MPO-Gd. Circles indicate location of heart grafts. Data are shown as mean ± SEM; n = 6 per group. (C) Magnified MR image of isograft 120 minutes after injection of MPO-Gd. Some focal bright signal reflects spin refreshment effects due to blood flow. Scale bars: 1 mm. (D) Allograft 120 minutes after injection of MPO-Gd. (E) Same data as in D, but the MPO-Gd signal was thresholded and pseudocolored in red. (F) Immunoreactive staining for MPO in the area that enhances in D and E. Scale bar: 20 μm. (G) Thresholded data of signal enhancement 120 minutes after injection of MPO-Gd show typical enhancement patterns. To highlight the more comprehensive sampling of imaging over the clinical standard, areas that would be accessible to heart biopsies, which are routinely taken from the right ventricular cavity, are color-coded in yellow. Red encodes foci of rejection that could not be reached with routine transvenous right ventricular biopsies.

Figure 5. Effects of clinically relevant immunosuppressive therapy 7 days after transplantation.

(A) MPO protein tissue level assessed by Western blotting and (B) biochemical determination of MPO tissue activity in isografts, allografts, and allografts of mice treated with prednisolone, azathioprine, and cyclosporine (Rx). (A) Data are shown as mean ± SEM; n = 3 per group. *P < 0.01 (C) Flow cytometric profiling of leukocytes in the same groups as above. (D and E) Quantitative immunoreactive staining for MPO, Ly-6C, macrophage (F4/80), and neutrophil marker (NIMP-R14) in the 3 groups. n = 3 mice per group, *P < 0.01. Scale bar: 10 μm. (F) Representative MPO-Gd MRI before and 120 minutes after injection. Circles indicate graft. Next to the graft, the bladder showed enhancement, indicating renal excretion of MPO-Gd. No enhancement was seen in the allograft treated with immunosuppressive therapy. (G) Change of CNR before injection versus 2 hours after injection in iso- and allografts of mice with and without treatment. n = 6 per group, *P < 0.05.

Figure 6. MPO-targeted MRI detects subtle transplant rejection.

(A) MR images acquired 2 hours after MPO-Gd in various groups and time points, H&E histology, and CD11b immunoreactive staining for myeloid cells and H&E histology. Scale bar: 25 μm. Arrows indicate enhancing regions after injection of MPO-Gd. (B) MR data acquired in mice treated with insufficient immunosuppression on day 7 after allograft. Pred, prednisolone. n = 4–6 per group. *P < 0.05 versus iso- and untreated allograft. (C) Serial imaging in isograft controls (n = 4) and single MHC class II mismatch (Bm12→B6, n = 7, *P < 0.05).

Figure 7. Translational potential of MPO as an imaging target for organ rejection.

(A) MPO protein levels measured by flow cytometry in human blood monocyte subsets. (B) MPO in biopsy samples from patients after heart transplantation. H&E stain and immunoreactive staining for MPO in representative sections from patient biopsies. Patient on left had no signs of rejection (grade 0) and no MPO⁺ cells, whereas patient on the right was graded 3R (severe rejection/high grade infiltrate) and showed MPO⁺ cells on immunoreactive staining. Scale bar: 20 μm. (C) Quantification of MPO⁺ cells in patients with no (ISHLT grade 0), mild (1R), and high grade (2/3R) cellular infiltration. Data are shown as mean ± SEM.