Chemokine Receptor 2-targeted Molecular Imaging in Pulmonary Fibrosis. A Clinical Trial

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is a progressive inflammatory lung disease without effective molecular markers of disease activity or treatment responses. Monocyte and interstitial macrophages that express the C-C motif CCR2 (chemokine receptor 2) are active in IPF and central to fibrosis.Objectives: To phenotype patients with IPF for potential targeted therapy, we developed 64Cu-DOTA-ECL1i, a radiotracer to noninvasively track CCR2+ monocytes and macrophages using positron emission tomography (PET).Methods: CCR2+ cells were investigated in mice with bleomycin- or radiation-induced fibrosis and in human subjects with IPF. The CCR2+ cell populations were localized relative to fibrotic regions in lung tissue and characterized using immunolocalization, single-cell mass cytometry, and Ccr2 RNA in situ hybridization and then correlated with parallel quantitation of lung uptake by 64Cu-DOTA-ECL1i PET.Measurements and Main Results: Mouse models established that increased 64Cu-DOTA-ECL1i PET uptake in the lung correlates with CCR2+ cell infiltration associated with fibrosis (n = 72). As therapeutic models, the inhibition of fibrosis by IL-1β blockade (n = 19) or antifibrotic pirfenidone (n = 18) reduced CCR2+ macrophage accumulation and uptake of the radiotracer in mouse lungs. In lung tissues from patients with IPF, CCR2+ cells concentrated in perifibrotic regions and correlated with radiotracer localization (n = 21). Human imaging revealed little lung uptake in healthy volunteers (n = 7), whereas subjects with IPF (n = 4) exhibited intensive signals in fibrotic zones.Conclusions: These findings support a role for imaging CCR2+ cells within the fibrogenic niche in IPF to provide a molecular target for personalized therapy and monitoring.Clinical trial registered with www.clinicaltrials.gov (NCT03492762).

Keywords: CCR2; macrophages; monocytes; positron emission tomography; pulmonary fibrosis.

Figures

Figure 1.

CCR2+ (C-C chemokine receptor 2–positive) cells localize to perifibrotic regions in bleomycin-induced lung fibrosis. C57BL/6 Ccr2gfp/+ mice were administered intranasal bleomycin, and lungs were assayed at the indicated day. (A) Representative images of CCR2-EGFP+ (enhanced green fluorescent protein–positive) cells, identified using an anti-EGFP antibody (detected by 3,3′-diaminobenzidine [DAB], brown, top), with serial tissue sections stained using trichrome (bottom). (B) Quantitation of CCR2-EGFP+ cells in lung sections (0.14 mm2/field; approximately 250 fields/lung; n = 3–5 mice/time point). (C) CCR2-EGFP+ cells in lung sections at each modified Ashcroft fibrosis score. Each cell count and fibrosis score are from the same field in serial sections. Significantly more CCR2-EGFP+ cells were in the regions with Ashcroft scores of 5–7 than those with scores of 0–4 at 14 and 28 days (D14/D28). (D) Representative pseudocolored density plots from the analysis of mass cytometry showing a shift in the percentage of CCR2-EGFP+ inflammatory monocytes (Ly6Chigh monos) and interstitial macrophages in single-cell preparations of whole mouse lungs on Days 0 and 28 after bleomycin. (E) Relative proportions of CCR2-EGFP+ myeloid cell populations at Day 28 after bleomycin identified by mass cytometry (n = 5 mice/d). Cells in D and E were identified using the gating strategy in Figure E2. The solid circles in B and C are values outside of the 5th and 95th percentiles. Significance in A and B was determined by Kruskal-Wallis with Dunn’s multiple comparison test. (A) Scale bars, 250 μm. BLM = bleomycin; MΦ = macrophage; MHC = major histocompatibility complex; NT = nontreated.

Figure 2.

Detection of 64Cu-DOTA-ECL1i uptake by positron emission tomography (PET)/computed tomography (CT) in bleomycin-induced lung fibrosis in mice. (A–D) Mice given intranasal bleomycin were injected with 64Cu-DOTA-ECL1i before dynamic PET/CT imaging on the indicated day. Representative transverse images are shown of (A) wild-type mice, (B) Ccr2 (chemokine receptor 2)-knockout mice (CCRgfp/gfp) at Day 14, and (C) wild-type mice in competitive receptor studies (blocking) at Day 14. (D) Lung uptake from A–C. Shown are the medians of n = 6–10 mice/d of mixed sexes compared with nontreated mice by Kruskal-Wallis with Dunn’s multiple comparison test. Day 14 knockout (n = 6) mice uptake and blocking (n = 6) were compared with Day 14 control conditions using the Mann-Whitney U test. (E) Example of regional cellularity and fibrosis (red borders) in a trichrome-stained tissue section compared with CT and PET/CT-fused coronal sections at Day 14 after bleomycin. (E) Scale bar, 1,000 μm. BL = blocking; BLM = bleomycin; KO = knockout; WT = wild type.

Figure 3.

Effect of IL-1β blockade and pirfenidone treatment on 64Cu-DOTA-ECL1i PET uptake in bleomycin-induced fibrosis. (A) Treatment scheme of wild-type mice administered intranasal bleomycin treated with intraperitoneal IgG or anti–IL-1β antibody three times weekly on Days 10–28. (B) Representative trichrome-stained lung sections at Day 28 (n = 3 IgG and 7 IL-1β mice/condition). (C) Ashcroft scores from lung sections (0.14 mm2/field; n = 360–500 fields/mouse lung; n = 3/group IgG and n = 4/group IL-1β). (D) Representative images of serial lung tissue sections stained with trichrome (top) and Ccr2 (C-C chemokine receptor 2) in situ hybridization (bottom) of indicated condition at Day 28. (E) Quantitation of Ccr2 in situ hybridization at Day 28 (1,400–1,900 fields/mouse lung; n = 3–4 mice/condition). (F) Representative 64Cu-DOTA-ECL1i positron emission tomography (PET)/computed tomography (CT) uptake in bleomycin-induced lung fibrosis in mice at Day 28 treated as indicated. (G) PET uptake in lungs after indicated treatment (n = 8–9 mice/condition). (H) Treatment scheme of mice administered bleomycin, treated with chow only or chow containing pirfenidone on Days 10–28. (I) Representative trichrome-stained lung sections at Day 28. (J) Ashcroft scores from lung sections (0.14 mm2/field; n = 33–36 fields/lung; n = 4 mice/condition). (K) Quantitation of Ccr2 in situ hybridization at Day 28 (1,600–1,900, 0.14 mm2 fields/mouse lung; n = 4 mice/condition). (L) Percentage of lung interstitial macrophages (SiglecF−/CD64+) in single-cell preparations of mouse lungs using mass cytometry (n = 7–8 mice/condition). (M) Representative PET/CT images at Day 28 treated as indicated. (N) PET uptake in lungs treated as indicated (n = 8–9 mice/condition). The solid circles in C, E, J, and K are values outside of the 5th and 95th percentiles. Significance was determined by Mann-Whitney U test for all data. Scale bars, (B and I) 1,000 μm and (D) 100 μm. ab = antibody; BLM = bleomycin; i.n. = intranasal; MΦ = macrophage; PFD = pirfenidone.

Figure 4.

Detection of CCR2+ (C-C chemokine receptor 2–positive) cells in lung tissue explanted from patients with pulmonary fibrosis (PF). Explanted lungs from patients with end-stage PF or from nonfibrotic lungs donated for research (donor). (A) Pretransplant chest computed tomography (CT) from a patient with idiopathic PF showing the region of explanted lung sampled (*) for serial tissue sections stained by trichrome and CCR2 antibody (green). Arrows indicate pleural surface. Yellow lines demarcate regions of high CCR2+ cells. (B) Representative serial tissues of donor lung stained as in A. (C) Total CCR2+ cells in samples (n = 4 donors and n = 11 PF). (D) CCR2+ cells from donor and PF lungs. Box plots represent a sample from each subject. Brackets mark fields with CCR2+ cell density above the 95th percentile of fields of donors. (E) Percentage of interstitial macrophages in lung tissues (median, n = 6 donors and n = 11 PF). (F) Representative comparison of trichrome, CCR2 staining, and 64Cu-DOTA-ECL1i autoradiography in serial human lung sections. Blocking studies confirm binding specificity (n = 9). Boxes show examples of paired areas analyzed for CCR2 immunofluorescent intensity compared with radioprobe binding determined by autoradiography in G. (G) Representative lung tissue with Spearman’s correlation of CCR2 immunostaining and corresponding 64Cu-DOTA-ECL1i pixel intensity in fields of high and low CCR2 immunofluorescence (boxes) from F (P = 0.0083; the full image of the tissue in F and G is shown in Figure E6A). For tissues from all subjects analyzed, the Spearman’s correlation range was 0.94–0.56 (all P < 0.05; n = 8 subjects). The solid circles in C and D are values outside of the 5th and 95th percentiles. In C and E, significance was determined by the Mann-Whitney U test. In A, B, E, and F the DAPI-stained nuclei are blue. Scale bars, (A and B) 500 μm and (F) 5 mm. MΦ = macrophage.

Figure 5.

Dosimetry testing of 64Cu-DOTA-ECL1i positron emission tomography (PET)/computed tomography (CT) imaging in healthy volunteers. (A) Representative coronal images of CT, maximum intensity projection PET, and PET/CT from a healthy volunteer obtained at the indicated time after a single intravenous injection with 64Cu-DOTA-ECL1i (n = 6). PET images show uptake in the liver, kidney, and bladder. Representative time–activity curves demonstrate (B) rapid blood clearance and (C) estimated renal clearance based on changes in bladder activity. HU = Hounsfield units; SUV = standard uptake value.

Figure 6.

64Cu-DOTA-ECL1i positron emission tomography (PET)/computed tomography (CT) imaging in patients with idiopathic pulmonary fibrosis (IPF). A healthy volunteer (vol) and subjects with IPF (n = 4) were injected with 64Cu-DOTA-ECL1i immediately before dynamic PET/CT imaging. (A) Representative sagittal plane CT, PET, and PET/CT-fused images show subpleural uptake (arrowheads). Regions of interest (ROIs) selected for analysis in healthy vol (blue) and subjects with IPF (nonfibrotic, yellow; fibrotic, orange) are indicated. (B) Representative coronal plane images of lungs demonstrating subpleural uptake. (C) Standard uptake values corrected for tissue density (tissue factor [TF]) in the indicated ROIs from A. Lines connect the corrected standard uptake values for the nonfibrotic and fibrotic ROIs for each subject with IPF. (D) Tissue background ratio corrected using the TF analyzed using the selected ROI of a healthy vol compared with subjects with IPF. HU = Hounsfield units; SUV = standard uptake value.