Introduction

Acute lung injury (ALI), clinically known as acute respiratory distress syndrome (ARDS), represents a life-threatening condition in seriously ill patients characterized by an acute onset of pulmonary infiltrates and impaired oxygenation. ARDS is an inflammatory driven disease resulting from direct (e.g., pneumonia) or indirect (e.g., sepsis or trauma) pulmonary insults leading to disruption of the alveolar-capillary barrier and, ultimately, impaired lung function [1]. Clinically, ARDS continues to significantly burden healthcare settings [2], particularly intensive care units, with the incidence of ARDS increasing many folds during the COVID-19 pandemic [3]. Despite our improved understanding on the risk factors that are associated with the development of ARDS, the diagnosis of ARDS remains clinical being based on a constellation of clinical, physiological, and radiographic findings, and we currently lack effective methods to predict patient’s outcomes and monitor treatment response.

A promising method for in vivo monitoring of molecular patterns of lung inflammation and response to therapy is positron emission tomography (PET). In particular, PET visualizes and quantifies ongoing molecular processes and therefore supplements the anatomical and structural information provided by other imaging modalities, such as computed tomography (CT). Additionally, PET has many advantages to study pulmonary inflammation in comparison to those of currently employed methods. For instance, PET, in contrast to lung biopsy or bronchoalveolar lavage (BAL), noninvasively visualizes lung inflammation without being subject to sampling bias [4]. Moreover, PET may detect molecular patterns of inflammation or resolution at an earlier stage than that of other imaging techniques, including CT [5]. Also, PET is a quantitative technique that is less affected by patient’s effort, which represents a major disadvantage of physiological tests, such as pulmonary function tests [6]. Further, PET can be tailored to monitor individual molecular targets or pathways of interest by the use of specific radiotracers. While much effort is currently being devoted to the development of PET tracers targeting specific aspects of the inflammatory response, the most widely available and studied radiotracer remains 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG).

Cellular uptake of [18F]FDG closely reflects glucose uptake as both [18F]FDG and glucose are internalized and processed by related cellular processes [7, 8]. [18F]FDG is transported into metabolically active cells via the glucose transporter (GLUT) family of proteins and is subsequently phosphorylated by hexokinases into [18F]FDG-6-phosphate, which is essentially irreversibly trapped intracellularly and cannot be further metabolized via glycolysis [9].

Although [18F]FDG is most extensively utilized for oncological purposes to detect the tumor-associated Warburg effect, the applications of [18F]FDG PET in inflammatory diseases have increased [10,11,12]. In the inflammatory environment, molecular signals and cascades alter immune cell metabolism in highly orchestrated ways, referred to as immunometabolism. One of the key cellular responses to inflammation is increased glucose metabolism [13]. Given that several cell types abundant in the acutely injured lung, such as neutrophils and macrophages, demonstrate increased influx and glucose metabolism in inflamed tissues, [18F]FDG PET is a clinically available method to sensitively and noninvasively visualize in vivo changes in glucose metabolism, ultimately a proxy for total inflammatory cell burden [5, 14, 15].

Clinically, [18F]FDG PET has been demonstrated to noninvasively monitor the underlying inflammatory processes in various pulmonary diseases. Lung uptake of [18F]FDG is increased in chronic obstructive pulmonary disease (COPD), cystic fibrosis, asthma, interstitial lung diseases, and ARDS [11, 14, 16,17,18,19,20,21]. Importantly, [18F]FDG PET provides clinically useful information about disease phenotypes and treatment response. In both COPD [17,18,19] and idiopathic pulmonary fibrosis [20], for instance, increased [18F]FDG uptake correlates with clinical markers of disease severity and indicates aggressive disease status. A separate study has demonstrated that both total and rate of [18F]FDG uptake are increased in patients with cystic fibrosis compared to healthy controls, and [18F]FDG uptake correlates with neutrophil content of BAL samples [21]. Increased lung uptake of [18F]FDG has also been reported in healthy volunteers instilled with intrabronchial endotoxin [11]. Interestingly, [18F]FDG PET has been shown to sensitively detect the lung-specific responses to therapies, including statins and recombinant activated protein C, in healthy volunteers receiving intrabronchial endotoxin [14]. While almost all immune and non-immune cells contribute to the total uptake of [18F]FDG in the tissues, neutrophils have been shown to have the highest ex vivo uptake of [3H]deoxyglucose among the cells recovered by BAL [11, 21].

Despite significant interest in exploring [18F]FDG as a method to noninvasively monitor lung inflammation, with a particular interest in improving our ability to molecularly endotype and stratify patients for improving clinical outcomes, there remain critical gaps in our knowledge that limit the interpretation of [18F]FDG PET and its utility for understanding lung diseases. Two key gaps include whether [18F]FDG uptake correlates with lung tissue expression of inflammatory biomarkers and if [18F]FDG can monitor the response to corticosteroids, as a clinically relevant anti-inflammatory therapy in ARDS. In this study, we demonstrated in a preclinical lipopolysaccharide (LPS)-induced ALI model that [18F]FDG PET noninvasively detected lung inflammation and response to dexamethasone therapy. Additionally, we showed that [18F]FDG uptake correlated with the expression of selected inflammatory proteins and activity of neutrophil elastase and glycolytic enzymes. We further showed that the total number of cells in the lungs, predominantly driven by changes in neutrophils, closely mirrored that of [18F]FDG uptake under different conditions.

Methods

Chemicals and Reagents

The major chemicals and reagents are listed in Supplemental Tables 1–5.

Mouse Model of Experimental Lung Injury

Animal experiments were performed using C57BL/6 J mice (9- to 12-week-old, Jackson Laboratory, strain #000664). Mice were administered intratracheally with 2.5 µg/g LPS from Escherichia coli O111:B4 in 60 µl of phosphate-buffered saline (PBS) to induce lung injury. Control mice were intratracheally injected with 60 µl of PBS. In experiments involving anti-inflammatory treatment, mice received two doses of dexamethasone (10 mg/kg in 500 µL of PBS) via intraperitoneal injections at 1 and 24 h following LPS instillation, whereas non-dexamethasone treated mice were given intraperitoneal PBS (500 µL) injections. Studies were performed in accordance with a protocol approved by the University of Pittsburgh Institutional Animal Care and Use Committee.

PET/CT and Quantification of [18F]FDG Uptake

Mice (n = 4 PBS, 8 LPS, and 8 LPS + Dexamethasone) were fasted the night prior to PET/CT. Two days after intratracheal LPS or PBS instillation, mice were injected intravenously with [18F]FDG (7.52 ± 0.03 MBq) while awake, and the mice remained awake until PET/CT acquisition. Mice were anesthetized with inhalational isoflurane and static PET (~ 5-min) and CT (180 projections, 140-ms exposure, 180° rotation, 80-kVp, 500-µA, field-of-view: 78.5 × 100-mm) were performed (Inveon, Siemens) ~ 60 min after radiotracer injection.

Regions of interest were drawn over the left and right lungs, and [18F]FDG uptake in the left and right lungs was averaged and quantified as the mean percentage of injected dose per milliliter of tissue (%ID/mLmean) and standard uptake value (SUVmean) (IRW software). The peripheral regions of the lungs were excluded from the regions of interest to minimize the effect of scattered radiation from [18F]FDG uptake by myocardium and bones. Following PET/CT, mice were euthanized, and radiotracer uptake was also quantified ex vivo by γ-counting (Wizard2, PerkinElmer) of harvested lungs. Decay-corrected [18F]FDG uptake values are reported.

Lung Homogenization

Following γ-counting, the lungs were frozen at -20 °C and stored for > 10 half-lives to allow [18F]FDG to fully decay. Then, the left lung was mechanically homogenized in 1 mL of distilled water. A 10 × cytokine lysis buffer (Table S2) was added to the lung homogenate (9 parts of lung homogenate: 1 part of 10 × cytokine lysis buffer, by volume). After incubating for 30 min at 4 °C, the lung homogenate was centrifuged (10,000 g for 20 min). The resulting supernatant was collected and stored at -80 °C until use.

Protein Expression Assays

The protein expression of select inflammatory markers in the lung homogenate was quantified using an enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s directions (Table S3).

Neutrophil Elastase Activity Assay

The neutrophil elastase activity in lung homogenates was determined as previously described, with minor modifications [22, 23]. Briefly, lung homogenate (10 µL) was incubated with 100 µM of a fluorogenic neutrophil elastase substrate (MeOSuc-AAPV-AMC, Millipore Sigma) in 50 µL assay buffer (100 mM Tris–HCl, 500 mM NaCl, pH = 8.0) at 37 °C. Over the course of 3 h, the fluorescence of each sample was measured every 10 min (Excitation = 380 nm, Emission = 460 nm) with a Synergy H4 Hybrid Microplate Reader (BioTek). The neutrophil elastase activity was calculated as the slope of the linear portion of the fluorescence versus time graph normalized to the average of that of the control mice.

Glycolytic Enzyme Activity Assays

The activity of hexokinase (HK) [24] and lactate dehydrogenase (LDH) [25] was measured using previously reported methods with optimization for use with lung homogenate. Briefly, lung homogenate samples (4.0 µL) were incubated at room temperature with 56.0 µL of the appropriate assay buffers (Tables S3 and S4). The absorbance of each sample, resulting from NADH or NADPH reduction of nitro blue tetrazolium chloride, was measured over 10 min at 1 min intervals at 490 nm [26] using a Synergy H4 Hybrid Microplate Reader (BioTek). The rate of enzymatic activities was determined as the slope of the linear portion of the absorbance versus time graph normalized to the average of that of the control mice.

Flow Cytometric Immunophenotyping of Murine Lungs

Separate groups of mice treated with LPS (N = 4), LPS + Dexamethasone (N = 4), or PBS (N = 4) were euthanized, and the pulmonary circulation was perfused with PBS via a right ventricular puncture. Both lungs were then minced with scissors and diluted with dissociation buffer (collagenase A, 1.0 mg/mL in PBS supplemented with 1 mM of Ca+2 and Mg+2). The lungs were enzymatically dissociated at 37 °C for 1 h with shaking. Then, the resulting cell suspensions were passed through 40-µm cell strainers. After red blood cell lysis, Precision Count Beads (50 µL) were added to each sample. After washing with PBS, cells were incubated with 1% bovine serum albumin (BSA) in PBS and treated first with mouse Fc block (2.0 µL per sample), followed by a mixture of antibodies (0.5 µL per antibody per sample) and DAPI (5.0 µL of 1 µg/mL per sample) for 0.5 h at 4 °C. Finally, cells were washed and fixed with 4% formalin for 0.5 h at room temperature. Flow cytometry was performed the following day using a LSR II Flow Cytometry (BD Biosciences) and analyzed using FlowJo software version 10.7.2 (BD Biosciences). A list of the antibodies used in this study is available in Table S1.

Statistics

Statistical analysis was performed with Prism 9 (GraphPad). The data are shown as the mean ± SEM. A one-way analysis of variance, followed by Fisher’s Exact post hoc test, was used to compare mean values in > 2 groups. Pearson correlations were used to determine the linear relationship between two variables. Statistical significance was considered as P < 0.05.

Results

Noninvasive Detection of ALI and Anti-inflammatory Response to Dexamethasone by [18F]FDG PET

At 1-h post-injection of [18F]FDG, we observed increased radiotracer uptake in the lungs of LPS-treated mice as compared to those of control mice (Fig. 1). The mice receiving LPS followed by dexamethasone demonstrated less lung [18F]FDG uptake than that of LPS-treated mice alone, but greater than that of the control mice. Quantification of PET-derived [18F]FDG uptake confirmed that LPS-treated mice demonstrated significantly increased tracer uptake over PBS-treated mice (by both PET-derived %ID/mLmean and SUVmean), which was significantly reduced by administration of dexamethasone (PET-derived %ID/mLmean) with a trend towards a significant decline as measured by SUVmean (Fig. 2A, B).

Fig. 1
figure 1

Representative [18F]FDG PET/CT images in LPS-induced ALI and its response to dexamethasone treatment. (A) Axial and (B) sagittal PET, CT, and coregistered PET/CT images for mice treated with PBS, LPS, or LPS followed by dexamethasone. Mice treated with LPS show focal regions of pulmonary consolidation and groundglass opacities on the CT, which demonstrate increased [18F]FDG uptake. Dexamethasone treatment decreases lung [18F]FDG uptake, although not to the level of control treated mice. High [18F]FDG uptake is seen in lung-adjacent tissues, including the heart, ribs, and brown fat. The lungs are outlined in white in the PET/CT overlaid images. The white letters indicate image direction: A = anterior, L = left, P = posterior, R = right. Abbreviations: PBS = phosphate-buffered saline, LPS = lipopolysaccharide, Dex = dexamethasone

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Fig. 2
figure 2

Quantification of [18F]FDG uptake in a preclinical LPS-induced ALI model confirms dexamethasone treatment response may be detected noninvasively by [18F]FDG PET. The in vivo uptake of [18F]FDG in the lungs was quantified by PET-derived (A) %ID/mLmean and (B) SUVmean. (C) Ex vivo γ-counting was performed on the lungs following completion of PET/CT. (D, E) Correlations between in vivo and ex vivo [18F]FDG uptake were calculated. N = 4 PBS, 8 LPS, and 8 LPS + Dex treated mice. Equal numbers of male (squares) and female (circles) mice were used per group. Colors: PBS = black, LPS = red, LPS + Dex = blue. Abbreviations: PBS = phosphate-buffered saline, LPS = lipopolysaccharide, Dex = dexamethasone

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Ex vivo Quantification of [18F]FDG Uptake by γ-counting Correlates Moderately with PET-derived %ID/mLmean, but Less Strongly with SUVmean

PET-derived quantification of the uptake of radiotracers is subject to inaccuracies resulting from lung-specific (e.g. respiratory motion and lung tissue density) [27, 28] and PET-specific (e.g. scatter, partial volume effect, and positron range) [29, 30] factors. Therefore, we determined the correlations between PET-derived [18F]FDG uptake (%ID/mLmean or SUVmean) in the lung and the uptake quantified by ex vivo γ-counting (%ID/g), the gold standard for quantification of radiotracer uptake by organs in preclinical studies, to address the accuracy of in vivo quantification of [18F]FDG. Similar to the [18F]FDG uptake determined by PET imaging, mice treated with LPS demonstrated significantly increased [18F]FDG uptake in the lungs, compared to that of control mice by ex vivo γ-counting (6.95% ± 0.48 vs. 2.43% ± 0.37, respectively; P < 0.0001) (Fig. 2C). Consistent with PET-derived quantification, mice receiving LPS followed by dexamethasone had an intermediate level of [18F]FDG uptake that was less than that of the LPS only treated mice (4.79% ± 0.19 vs. 6.95% ± 0.48, respectively; P = 0.0004) yet greater than that of the control mice (4.79% ± 0.19 vs. 2.43% ± 0.37, respectively; P = 0.0011). Importantly, there was a moderate correlation between in vivo PET-derived %ID/mLmean and ex vivo γ-counting-derived (%ID/g) [18F]FDG lung uptake (R2 = 0.53; P = 0.0003) (Fig. 2D, E). However, the correlation between in vivo PET-derived SUVmean and ex vivo γ-counting-derived (%ID/g) [18F]FDG uptake was less strong (R2 = 0.24; P = 0.027).

[18F]FDG Uptake Correlates with Inflammatory Biomarkers and the Activity of Glycolytic Enzymes

One of the hallmarks of experimental ALI is the inflammatory response that is characterized by an increase in cytokines and chemokines [31]. We tested whether increased [18F]FDG lung uptake in ALI correlates with the abundance of select inflammatory proteins. We found that LPS treatment significantly increased the pulmonary expression of pro-inflammatory cytokines and chemokines [1, 32,33,34,35], including interleukin 1-beta (IL-1β), interferon gamma (IFN-γ), interleukin 6 (IL-6), and CXC motif chemokine ligand 1 (CXCL1), versus that of control treated mice (Fig. 3A). Additionally, dexamethasone reduced the expression of these cytokines and chemokines to an intermediate level between that of the LPS-treated and control mice. We further established that lung uptake of [18F]FDG, as measured with in vivo %ID/mLmean or SUVmean or ex vivo %ID/g, generally correlated with the expression of established inflammatory markers (Fig. 3B). In particular, the ex vivo [18F]FDG uptake most strongly correlated with inflammatory markers IL-1β and CXCL1.

Fig. 3
figure 3

PET-derived [18F]FDG uptake correlates with protein expression of inflammatory markers. (A) The protein expression of select markers was determined with an enzyme-linked immunosorbent assay following PET/CT. (B) Expression of inflammatory cytokines/chemokines was correlated with PET-derived (Top: %ID/mLmean or Middle: SUVmean) and γ-counter-derived (Bottom) [18F]FDG uptake. N = 4 PBS, 8 LPS, and 8 LPS + Dex treated mice. Equal numbers of male (squares) and female (circles) mice were used per group. Abbreviations: PBS = phosphate buffered saline, LPS = lipopolysaccharide, Dex = dexamethasone

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Recruitment and activation of neutrophils are key features of the initial inflammatory phase of ALI [31], which are mediated by several chemokines, including CXCL1. We therefore tested if [18F]FDG uptake correlated with neutrophil elastase activity in lung homogenate, as a measure for the total burden of neutrophil activation in the lungs [23, 36]. As expected, LPS induced a significant increase in lung neutrophil elastase activity, versus that of control mice, that was decreased upon dexamethasone treatment (Fig. 4A). Although ex vivo γ-counting-derived [18F]FDG uptake correlated with neutrophil elastase activity (R2 = 0.43, P = 0.0017), the correlations were weaker with PET-derived quantification of [18F]FDG uptake (%ID/mLmean: R2 = 0.18, P = 0.064 and SUVmean: R2 = 0.28, P = 0.017) (Fig. 4B).

Fig. 4
figure 4

Pulmonary [18F]FDG uptake correlates with neutrophil elastase activity. (A) Neutrophil elastase activity was determined in lung homogenates following PET/CT. The total neutrophil elastase activity is increased following intratracheal instillation of LPS and decreases nearly to baseline with dexamethasone treatment. The neutrophil elastase activity of LPS and LPS + Dex groups was normalized to the average of the controls. (B) Neutrophil elastase activity was correlated with PET-derived (Left: %ID/mLmean or Middle: SUVmean) or γ-counter-derived (Right) [18F]FDG uptake. N = 4 PBS, 8 LPS, and 8 LPS + Dex treated mice. Equal numbers of male (squares) and female (circles) mice were used per group. Abbreviations: PBS = phosphate buffered saline, LPS = lipopolysaccharide, Dex = dexamethasone

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As several inflammatory stimuli inhibit the tricarboxylic acid cycle, many leukocytes in inflammatory environments rely on glycolysis for energy production and proper inflammatory functions [37,38,39,40,41]. Further, a switch to glycolysis with an associated increase in lactate production represents a validated metabolic alteration and poor clinical prognostic factor in ARDS [42,43,44,45,46,47,48,49]. We therefore examined whether [18F]FDG uptake correlates with pulmonary glycolytic activity of HK and LDH. We focused on HK and LDH as they represent important steps in glycolysis; HK traps glucose and [18F]FDG intracellularly via phosphorylation whereas LDH catalyzes the critical final step of glycolysis to regenerate NAD + to sustain high levels of glycolysis. Following LPS treatment, we found that the relative activity of HK and LDH were both significantly increased over that of controls (Fig. 5A). Interestingly, dexamethasone decreased HK and LDH activities to the levels of control mice. Further, the HK and LDH activity both generally correlated with [18F]FDG uptake, although most strongly with ex vivo γ-counting-derived measurements (Fig. 5B).

Fig. 5
figure 5

[18F]FDG uptake correlates with the activity of glycolytic enzymes. (A) The activity of hexokinase (HK) and lactate dehydrogenase (LDH) in lung homogenates was measured following PET/CT. The total lung activity of both enzymes was significantly increased following administration of LPS. Anti-inflammatory treatment by dexamethasone reduced the activity of HK and LDH nearly to that of controls. Enzymatic activity was normalized to that of the average of the controls. (B) HK and LDH activities were correlated with PET-derived (Left: %ID/mLmean or Middle: SUVmean) or γ-counter-derived (Right) [18F]FDG uptake. N = 4 PBS, 8 LPS, and 8 LPS + Dex treated mice. Equal numbers of male (squares) and female (circles) mice were used per group. Abbreviations: PBS = phosphate-buffered saline, LPS = lipopolysaccharide, Dex = dexamethasone

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Neutrophil Burden in the Lungs Mirrors the Observed Pattern of [18F]FDG Uptake in LPS-induced ALI and Response to Anti-inflammatory Treatment

To determine changes in pulmonary immune cell populations in response to LPS administration with or without dexamethasone treatment, we first determined the absolute cell count for individual cell types harvested from the lungs (Fig. 6A) using a flow cytometry gating strategy provided in Fig. S1. Following LPS-treatment, we detected significantly increased numbers of lung neutrophils (e.g., ~ 60-fold increase) versus that of control mice (P = 0.0008) (Fig. 6A). However, LPS induced only modest increases in the number of several other lung immune cell populations, including interstitial and monocyte-derived macrophages. We found that dexamethasone treatment reduced the number of infiltrating neutrophils without affecting the abundance of other cell populations. Additionally, we observed minimal changes to the number of lymphoid cells (B-cells, natural killer cells, and T-cells), monocytes (Ly6Chi and Ly6Clo), dendritic cells (CD11bhi and CD11blo), eosinophils, and CD45neg non-immune cells after treatment with LPS or dexamethasone versus that of controls.

Fig. 6
figure 6

Neutrophils are the most abundant cell type during LPS-induced ALI as measured by flow cytometry. (A) The cell count was quantified for mice treated with intratracheal LPS or LPS + Dex vs. control. (B) The relative contribution of individual cell types to the total recovered cells from the lungs of mice following treatment with LPS and/or dexamethasone versus control, as visualized with a stacked-bar graph. Abbreviations: NK cells = natural killer cells; aMφ = alveolar macrophage; iMφ = interstitial macrophage; MDMφ = monocyte-derived macrophage; CD11bhi DC = CD11bhi dendritic cells; CD11blo DC = CD11blo dendritic cells; Eosin = eosinophils; CD45neg cells = non-immune cells. N = 4 PBS, 4 LPS, and 4 LPS + Dex treated mice. Equal numbers of male and female mice were used per group. Abbreviations: PBS = phosphate-buffered saline, LPS = lipopolysaccharide, Dex = dexamethasone

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We next calculated the relative contributions of individual cell types to the total pulmonary cell counts under different treatment conditions (Fig. 6B). Unsurprisingly, LPS-treated mice exhibited the highest number of total lung cells, with neutrophils accounting for over half of all cells in the lungs of mice with LPS-induced ALI. Dexamethasone therapy decreased the number of lung leukocytes largely due to a decrease in the number of neutrophils. By contrast, neutrophils represented only 2% of all cells in the control lungs. As the changes in CD45neg cells and other leukocyte populations were relatively minor compared to that of the neutrophils, the change in total cell count was driven primarily by neutrophils. Furthermore, we observed the total cell count measured by flow cytometry in different experimental groups (Fig. 6B) closely mirrored that of [18F]FDG uptake under the same treatment conditions (Fig. 2). Unsurprisingly, CD45neg cells represent a significant fraction of total lung cells under all conditions and are presumably a major contributor to [18F]FDG uptake in healthy lungs.

Discussion

In this study, we demonstrate that [18F]FDG uptake is increased in LPS-induced ALI and noninvasively detects the response to a clinically relevant anti-inflammatory therapy, dexamethasone. We found that [18F]FDG uptake during ALI with or without dexamethasone treatment correlates with the degree of inflammatory cytokine/chemokine expression and the activity of neutrophil elastase and glycolytic enzymes. We further found that [18F]FDG uptake closely mirrors the pattern of pulmonary neutrophil accumulation during ALI, the dominant immune cell population in this model. Thus, [18F]FDG PET is effective in monitoring ongoing and resolving lung inflammation through its correlations with lung specific expression of inflammatory biomarkers and active neutrophils.

While the pathophysiology of ARDS/ALI is complex, we demonstrate that [18F]FDG PET noninvasively and directly monitors specific aspects of the inflammatory response in the lung that are difficult to obtain via other available techniques. We show that [18F]FDG uptake correlates with pathogenically relevant and therapeutically targetable biomarkers expressed in lung tissue, which may prove advantageous over measurement of serum biomarkers that may reflect systemic rather than lung specific inflammatory processes. Importantly, we found that [18F]FDG uptake monitors the anti-inflammatory response to dexamethasone therapy, as confirmed by correlations between reduced radiotracer uptake with decreased expression of inflammatory mediators, including cytokines, chemokines, and proteases. Furthermore, [18F]FDG PET more accurately visualizes the response to anti-inflammatory treatment compared to that of CT, as demonstrated by reduced [18F]FDG uptake in regions of CT-defined pulmonary airspace opacification following steroid therapy.

Despite the widespread use of and research on [18F]FDG for imaging inflammation, there remain biologic and technological limitations that may hinder interpretability of [18F]FDG for monitoring lung inflammation. For example, both clinical and preclinical studies have shown that all metabolically active cells metabolize [18F]FDG, including the liver, ribs, heart, and brown fat [50,51,52,53]. Due to the positron range and scattered radiation, the high radiotracer uptake in extra-pulmonary tissues, enhanced by the systemic inflammation induced by LPS, appears to spillover into the lungs, and leads to overestimation of the true extent of pulmonary [18F]FDG uptake by PET in regions adjacent to organs with high [18F]FDG uptake. In preclinical experiments, the combination of respiratory motion from non-respiratory gated scans and small subject size further enhances the undesirable impact of the relatively high positron range and scattered radiation, ultimately leading to inaccurate assessments of pulmonary radiotracer uptake. These limitations may account for the moderate correlation between ex vivo γ-counting (%ID/g) derived [18F]FDG uptake and in vivo PET %ID/mLmean and fair correlation between ex vivo γ-counting (%ID/g) derived [18F]FDG uptake and in vivo PET SUVmean. The lower correlation strength of SUVmean with γ-counting-derived uptake values may result from the weight loss in LPS-treated mice that lowers the SUVmean [54,55,56,57]. As the %ID/mL does not account for weight changes, we observed a stronger correlation between PET-derived %ID/mLmean and γ-counting derived %ID/g. Notably, the correlations between [18F]FDG uptake and biological markers in our study were generally strongest when tracer uptake was assessed by ex vivo γ-counting, consistent with the inaccuracies of in vivo PET-derived measurements. While the larger size of humans and implementation of respiratory gating in clinical scans reduce the above mentioned effects, the non-specific [18F]FDG uptake by the heart, liver, and ribs may obscure inflammatory processes in the lung periphery and subpleural space.

Although [18F]FDG PET is the most popular molecular imaging approach for assessment of inflammation, there remains a clear need to develop new radiotracers targeting specific aspects of the inflammatory process. In particular, our study demonstrates that [18F]FDG uptake is likely driven by abundant, metabolically active cell types, e.g., neutrophils in the case of ALI [4, 10, 11, 58, 59], with probable contributions from lung parenchymal cells. Additionally, prior studies have shown that neutrophils contribute to most [3H]deoxyglucose uptake among the cells recovered from BAL of humans intrabronchially administered with LPS [11, 21]. To address the non-specificity of [18F]FDG, selective PET agents have been developed to study specific inflammatory pathways, particularly monocyte/macrophage-driven inflammation. For instance, the PET tracer [64Cu]DOTA-ECL1i is reported to target CCR2-expressing monocytes [60, 61], which have been demonstrated to be relevant in pulmonary inflammation and noninvasively monitors response to immunotherapies. Additionally, we have recently reported that [64Cu]NODAGA-CG34, a PET tracer that selectively binds to chemokine-like receptor-1 (CMKLR1), detects the accumulation of monocyte-derived macrophages in the lungs of mice with ALI [62]. Other emerging molecular imaging targets include, α4β1 integrin [63], leukocyte receptor CD11b [64], folate receptor-β [65], and translocator protein receptor [66], which have been successfully used for noninvasive detection of lung inflammation in ALI.

There remain several limitations of our current study. First, as previously mentioned, the high degree of [18F]FDG uptake in lung-adjacent tissues results in the spillover of radiotracer signal into the lungs, as measured by PET. To minimize the unwanted contribution from these non-lung organs to the lung [18F]FDG uptake, we drew our regions of interest to include a few millimeter margins between the lung and adjacent organs, leading to underestimation of lung radiotracer uptake. Another limitation of our study is that we measured a relatively limited number of inflammatory and glycolytic markers with which to correlate [18F]FDG uptake. We chose to measure these chemokines, cytokines, and enzymes as they are common biomarkers characterizing the inflammatory response observed in experimental ALI [31]. Additionally, decreased blood flow to areas of lung inflammation due to hypoxia-induced vasoconstriction [67] is expected to affect the observed [18F]FDG uptake. Kinetic analysis of [18F]FDG uptake along with perfusion PET imaging allows for a more accurate measurement of the rate of [18F]FDG uptake. However, the static PET design of our approach is aligned with our primary goal to determine the utility of the [18F]FDG PET according to the currently performed clinical protocols to monitor lung inflammation and response to therapy.

In conclusion, [18F]FDG PET may be useful in monitoring ongoing pulmonary inflammation and therapeutic response. Additionally, our data along with the previous publications [11, 21] suggest that [18F]FDG uptake is mostly driven by the metabolic activity of neutrophils in this preclinical model of ALI, and is less reflective of other specific subsets of immune cells. While our results should encourage additional work into better understanding the biological correlations of [18F]FDG uptake, there remains a clear need to develop new radiotracers targeting specific aspects of the inflammatory process and better understand the biologic correlates of molecular imaging.