PET Imaging of Translocator Protein as a Marker of Malaria-Associated Lung Inflammation

Abstract

Malaria-associated acute respiratory distress syndrome (MA-ARDS) is a severe complication of malaria that occurs despite effective antimalarial treatment. Currently, noninvasive imaging procedures such as chest X-rays are used to assess edema in established MA-ARDS, but earlier detection methods are needed to reduce morbidity and mortality. The early stages of MA-ARDS are characterized by the infiltration of leukocytes, in particular monocytes/macrophages; thus, monitoring of immune infiltrates may provide a useful indicator of early pathology. In this study, Plasmodium berghei ANKA-infected C57BL/6 mice, a rodent model of MA-ARDS, were longitudinally imaged using the 18-kDa translocator protein (TSPO) imaging agent [¹⁸F]FEPPA as a marker of macrophage accumulation during the development of pathology and in response to combined artesunate and chloroquine diphosphate (ART+CQ) therapy. [¹⁸F]FEPPA uptake was compared to blood parasitemia levels and to levels of pulmonary immune cell infiltrates by using flow cytometry. Infected animals showed rapid increases in lung retention of [¹⁸F]FEPPA, correlating well with increases in blood parasitemia and pulmonary accumulation of interstitial inflammatory macrophages and major histocompatibility complex class II (MHC-II)-positive alveolar macrophages. Treatment with ART+CQ abrogated this increase in parasitemia and significantly reduced both lung uptake of [¹⁸F]FEPPA and levels of macrophage infiltrates. We conclude that retention of [¹⁸F]FEPPA in the lungs is well correlated with changes in blood parasitemia and levels of lung-associated macrophages during disease progression and in response to ART+CQ therapy. With further development, TSPO biomarkers may have the potential to accurately assess the early onset of MA-ARDS.

Keywords: PET; PbA; lung; macrophages; malaria.

FIG 1

(A) Timeline showing drug-dosing regimen. C57BL/6 mice were infected with 10⁶ P. berghei ANKA organisms on day 0. Animals were treated with combined artesunate (30 mg/kg) and chloroquine diphosphate (120 mg/kg) therapy or saline vehicle on days 5 to 11 inclusive (i.p., twice a day [b.i.d.]). Blood sampling was performed daily from days 5 to 11 and subsequently on days 14, 17, and 21 for parasite quantification. In vivo imaging was performed at 5, 6, and 7 dpi for the untreated group (n = 6) and at 7, 14, and 21 dpi for the treated group (n = 6). (B) Parasitemia levels in P. berghei ANKA-infected mice that were left untreated (n = 10) (filled circles) or treated with ART+CQ (n = 10) (open circles). Asterisks indicate significant differences (**, P < 0.01; ***, P < 0.001; ****, P < 0.001) between the log-transformed parasitemia levels in the two groups as determined by an unpaired Student t test. Data are shown as the mean log percentages of parasitemia ± SD.

FIG 2

Representative standardized PET-CT maximum-intensity projection images for the assessment of lung uptake of [¹⁸F]FEPPA in mice on days 5, 6, and 7 after P. berghei ANKA infection and after 2 days of combined ART+CQ treatment. Yellow arrows indicate lung uptake, and white arrows indicate kidney clearance.

FIG 3

In vivo assessment of lung uptake of [¹⁸F]FEPPA from PET-CT defined volumes of interest (VOI) from groups of P. berghei ANKA-infected C57BL/6 mice (n = 6). (A) Lung uptake of [¹⁸F]FEPPA increased at 6 dpi (filled bar), reaching the maximum at 7 dpi. Asterisks indicate significant differences (***, P < 0.001) from background results for naive mice (open bar) by an unpaired Student t test. Data are shown as mean percentages of the ID per gram ± SD. (B) Treatment with combined ART+CQ led to significant decreases in [¹⁸F]FEPPA uptake in the lung 2 days after the initiation of treatment (day 7) (dark shaded bar) compared to uptake in untreated P. berghei ANKA-infected animals (filled bar). [¹⁸F]FEPPA uptake levels similar to background levels in naive mice (open bar) were observed 3 days (day 14) (medium shaded bar) and 10 days (day 21) (light shaded bar) after the cessation of treatment. Symbols indicate significant differences by one-way ANOVA (Kruskal-Wallis test with a Bonferroni posttest) in multiple comparisons against data for untreated (*, P < 0.05; **, P < 0.01; ***, P < 0.001) and treated ($, P < 0.05) P. berghei ANKA-infected animals on day 7. Data are shown as median percentages of ID per gram ± interquartile range (IQR). (C) Correlation of lung uptake of [¹⁸F]FEPPA with parasitemia over the time course studied (Pearson’s r² = 0.904).

FIG 4

Assessment of myeloid cells in the lungs of infected mice by flow cytometry. (A) Levels of inflammatory macrophages increased significantly after P. berghei ANKA infection at 6 dpi (light shaded bar) and 7 dpi (filled bar) over background levels in naive animals (open bar). Treatment with combined ART+CQ led to significant decreases in levels of inflammatory macrophages 2 days after the initiation of treatment (day 7) (dark shaded bar) and 10 days after the cessation of treatment (day 21) (medium shaded bar) (n = 5). Asterisks indicate significant differences (**, P < 0.01; ***, P < 0.001) by one-way ANOVA. Data are shown as mean percentages of CD45⁺ cells ± SD. NS, no significant differences from results for naive animals. (B) Levels of MHC-positive alveolar macrophages increased significantly at 6 dpi (light shaded bar) and 7 dpi (filled bar) over background levels in naive animals (open bar).Treatment with combined ART+CQ led to significant decreases in MHC-positive alveolar macrophages 2 days after the initiation of treatment (day 7) (dark shaded bar) and 10 days after the cessation of treatment (day 21) (medium shaded bar) (n = 6). Asterisks indicate significant differences (*, P < 0.05; **, P < 0.01) by one-way ANOVA. Data are shown as mean percentages of CD45⁺ cells ± SD. NS, no significant differences from results for naive animals.