Noninvasive assessment of the lung inflammation-fibrosis axis by targeted imaging of CMKLR1.

Precision management of fibrotic lung diseases is challenging due to their diverse clinical trajectories and lack of reliable biomarkers for risk stratification and therapeutic monitoring. Here, we validated the accuracy of CMKLR1 as an imaging biomarker of the lung inflammation-fibrosis axis. By analyzing single-cell RNA sequencing datasets, we demonstrated CMKLR1 expression as a transient signature of monocyte-derived macrophages (MDMφ) enriched in patients with idiopathic pulmonary fibrosis (IPF). Consistently, we identified MDMφ as the major driver of the uptake of CMKLR1-targeting peptides in a murine model of bleomycin-induced lung fibrosis. Furthermore, CMKLR1-targeted positron emission tomography in the murine model enabled quantification and spatial mapping of inflamed lung regions infiltrated by CMKLR1-expressing macrophages and emerged as a robust predictor of subsequent lung fibrosis. Last, high CMKLR1 expression by bronchoalveolar lavage cells identified an inflammatory endotype of IPF with poor survival. Our investigation supports the potential of CMKLR1 as an imaging biomarker for endotyping and risk stratification of fibrotic lung diseases.

Targeted imaging of very late antigen-4 for noninvasive assessment of lung inflammation-fibrosis axis.

BACKGROUND: The lack of noninvasive methods for assessment of dysregulated inflammation as a major driver of fibrosis (i.e., inflammation-fibrosis axis) has been a major challenge to precision management of fibrotic lung diseases. Here, we determined the potential of very late antigen-4 (VLA-4)-targeted positron emission tomography (PET) to detect inflammation in a mouse model of bleomycin-induced fibrotic lung injury. METHOD: Single time-point and longitudinal VLA-4-targeted PET was performed using a high-affinity peptidomimetic radiotracer, 64Cu-LLP2A, at weeks 1, 2, and 4 after bleomycin-induced (2.5 units/kg) lung injury in C57BL/6J mice. The severity of fibrosis was determined by measuring the hydroxyproline content of the lungs and expression of markers of extracellular matrix remodeling. Flow cytometry and histology was performed to determine VLA-4 expression across different leukocyte subsets and their spatial distribution. RESULTS: Lung uptake of 64Cu-LLP2A was significantly elevated throughout different stages of the progression of bleomycin-induced injury. High lung uptake of 64Cu-LLP2A at week-1 post-bleomycin was a predictor of poor survival over the 4-week follow up, supporting the prognostic potential of 64Cu-LLP2A PET during the early stage of the disease. Additionally, the progressive increase in 64Cu-LLP2A uptake from week-1 to week-4 post-bleomycin correlated with the ultimate extent of lung fibrosis and ECM remodeling. Flow cytometry revealed that LLP2A binding was restricted to leukocytes. A combination of increased expression of VLA-4 by alveolar macrophages and accumulation of VLA-4-expressing interstitial and monocyte-derived macrophages as well as dendritic cells was noted in bleomycin-injured, compared to control, lungs. Histology confirmed the increased expression of VLA-4 in bleomycin-injured lungs, particularly in inflamed and fibrotic regions. CONCLUSIONS: VLA-4-targeted PET allows for assessment of the inflammation-fibrosis axis and prediction of disease progression in a murine model. The potential of 64Cu-LLP2A PET for assessment of the inflammation-fibrosis axis in human fibrotic lung diseases needs to be further investigated.

2-deoxy-2-[18F]fluoro-D-glucose Positron Emission Tomography to Monitor Lung Inflammation and Therapeutic Response to Dexamethasone in a Murine Model of Acute Lung Injury.

PURPOSE: To image inflammation and monitor therapeutic response to anti-inflammatory intervention using 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET) in a preclinical model of acute lung injury (ALI). PROCEDURES: Mice were intratracheally administered lipopolysaccharide (LPS, 2.5 mg/kg) to induce ALI or phosphate-buffered saline as the vehicle control. A subset of mice in the ALI group received two intraperitoneal doses of dexamethasone 1 and 24 h after LPS. [18F]FDG PET/CT was performed 2 days after the induction of ALI. [18F]FDG uptake in the lungs was quantified by PET (%ID/mLmean and standardized uptake value (SUVmean)) and ex vivo γ-counting (%ID/g). The severity of lung inflammation was determined by quantifying the protein level of inflammatory cytokines/chemokines and the activity of neutrophil elastase and glycolytic enzymes. In separate groups of mice, flow cytometry was performed to estimate the contribution of individual immune cell types to the total pulmonary inflammatory cell burden under different treatment conditions. RESULTS: Lung uptake of [18F]FDG was significantly increased during LPS-induced ALI, and a decreased [18F]FDG uptake was observed following dexamethasone treatment to an intermediate level between that of LPS-treated and control mice. Protein expression of inflammatory biomarkers and the activity of neutrophil elastase and glycolytic enzymes were increased in the lungs of LPS-treated mice versus those of control mice, and correlated with [18F]FDG uptake. Furthermore, dexamethasone-induced decreases in cytokine/chemokine protein levels and enzyme activities correlated with [18F]FDG uptake. Neutrophils were the most abundant cells in LPS-induced ALI, and the pattern of total cell burden during ALI with or without dexamethasone therapy mirrored that of [18F]FDG uptake. CONCLUSIONS: [18F]FDG PET noninvasively detects lung inflammation in ALI and its response to anti-inflammatory therapy in a preclinical model. However, high [18F]FDG uptake by bone, brown fat, and myocardium remains a technical limitation for quantification of [18F]FDG in the lungs.

Molecular imaging of chemokine-like receptor 1 (CMKLR1) in experimental acute lung injury.

The lack of techniques for noninvasive imaging of inflammation has challenged precision medicine management of acute respiratory distress syndrome (ARDS). Here, we determined the potential of positron emission tomography (PET) of chemokine-like receptor-1 (CMKLR1) to monitor lung inflammation in a murine model of lipopolysaccharide-induced injury. Lung uptake of a CMKLR1-targeting radiotracer, [64Cu]NODAGA-CG34, was significantly increased in lipopolysaccharide-induced injury, correlated with the expression of multiple inflammatory markers, and reduced by dexamethasone treatment. Monocyte-derived macrophages, followed by interstitial macrophages and monocytes were the major CMKLR1-expressing leukocytes contributing to the increased tracer uptake throughout the first week of lipopolysaccharide-induced injury. The clinical relevance of CMKLR1 as a biomarker of lung inflammation in ARDS was confirmed using single-nuclei RNA-sequencing datasets which showed significant increases in CMKLR1 expression among transcriptionally distinct subsets of lung monocytes and macrophages in COVID-19 patients vs. controls. CMKLR1-targeted PET is a promising strategy to monitor the dynamics of lung inflammation and response to anti-inflammatory treatment in ARDS.

Divergence of acetate uptake in proinflammatory and inflammation-resolving macrophages: implications for imaging atherosclerosis.

BACKGROUND: Metabolic divergence of macrophages polarized into different phenotypes represents a mechanistically relevant target for non-invasive characterization of atherosclerotic plaques using positron emission tomography (PET). Carbon-11 (11C)-labeled acetate is a clinically available tracer which accumulates in atherosclerotic plaques, but its biological and clinical correlates in atherosclerosis are undefined. METHODS AND RESULTS: Histological correlates of 14C-acetate uptake were determined in brachiocephalic arteries of western diet-fed apoE-/- mice. The effect of polarizing stimuli on 14C-acetate uptake was determined by proinflammatory (interferon-γ + lipopolysaccharide) vs inflammation-resolving (interleukin-4) stimulation of murine macrophages and human carotid endarterectomy specimens over 2 days. 14C-acetate accumulated in atherosclerotic regions of arteries. CD68-positive monocytes/macrophages vs smooth muscle actin-positive smooth muscle cells were the dominant cells in regions with high vs low 14C-acetate uptake. 14C-acetate uptake progressively decreased in proinflammatory macrophages to 25.9 ± 4.5% of baseline (P < .001). A delayed increase in 14C-acetate uptake was induced in inflammation-resolving macrophages, reaching to 164.1 ± 21.4% (P < .01) of baseline. Consistently, stimulation of endarterectomy specimens with interferon-γ + lipopolysaccharide decreased 14C-acetate uptake to 66.5 ± 14.5%, while interleukin-4 increased 14C-acetate uptake to 151.5 ± 25.8% compared to non-stimulated plaques (P < .05). CONCLUSIONS: Acetate uptake by macrophages diverges upon proinflammatory and inflammation-resolving stimulation, which may be exploited for immunometabolic characterization of atherosclerosis.

Imaging Immunometabolism in Atherosclerosis.

Over the past decade, there has been a growing recognition of the links between intracellular metabolism and immune cell activation, that is, immunometabolism, and its consequences in atherogenesis. However, most immunometabolic investigations have been conducted in cultured cells through pharmacologic or genetic manipulations of selected immunologic or metabolic pathways, limiting their extrapolation to the complex microenvironment of plaques. In vivo metabolic imaging is ideally situated to address this gap and to determine the clinical implications of immunometabolic alterations for diagnosis and management of patients. Indeed, 18F-FDG has been widely used in clinical studies with promising results for risk stratification of atherosclerosis and monitoring the response to therapeutic interventions, though the biologic basis of its uptake in plaques has been evolving. Herein, we describe recent advances in understanding of immunometabolism of atherosclerosis with an emphasis on macrophages, and we review promising metabolic imaging approaches using 18F-FDG and other PET radiotracers.

Identification of a novel spinal nociceptive-motor gate control for Aδ pain stimuli in rats.

Physiological responses to nociceptive stimuli are initiated within tens of milliseconds, but the corresponding sub-second behavioral responses have not been adequately explored in awake, unrestrained animals. A detailed understanding of these responses is crucial for progress in pain neurobiology. Here, high-speed videography during nociceptive Aδ fiber stimulation demonstrated engagement of a multi-segmental motor program coincident with, or even preceding, withdrawal of the stimulated paw. The motor program included early head orientation and adjustments of the torso and un-stimulated paws. Moreover, we observed a remarkably potent gating mechanism when the animal was standing on its hindlimbs and which was partially dependent on the endogenous opioid system. These data reveal a profound, immediate and precise integration of nociceptive inputs with ongoing motor activities leading to the initiation of complex, yet behaviorally appropriate, response patterns and the mobilization of a new type of analgesic mechanism within this early temporal nociceptive window.

Photo-degradation of 2,4-dinitroanisole (DNAN): An emerging munitions compound.

The US military is developing insensitive munitions (IM) that are less sensitive to shock and high temperatures to minimize unintentional detonations. DNAN (2,4-dinitroanisole) is one of the main ingredients of these IM formulations. During live-fire training, chunks of IM formulations are scattered by partial detonations and, once on the soil, they weather and dissolve. DNAN changes color when exposed to sunlight suggesting that it photodegrades into other compounds. We investigated the photo-degradation of DNAN both as a pure solid and as part of solid IM formulations, IMX101, IMX104 and PAX21. The concentrations of degradation products found were small, <1%, relative to DNAN concentrations. We saw transient peaks in the chromatograms indicating intermediate, unstable products but we consistently found methoxy nitrophenols and methoxy nitroanilines. We also found one unknown in most of the samples and other unknowns less frequently.

Synthesis of a Masked 2,3-Diaminoindole.

A three-step synthesis of masked 2,3-diaminoindole 1 from 2-iodo-3-nitro-1-(phenylsulfonyl)indole (2) has been developed. Treatment of 1 with trifluoroacetic acid generates the unstable 2,3-diamino-1-(phenylsulfonyl)indole (3), which can be trapped with α-dicarbonyl compounds to afford 5H-pyrazino[2,3-b]indoles 7-10.

Triple Benzannulation of Naphthalene via a 1,3,6-Naphthotriyne Synthetic Equivalent. Synthesis of Dibenz[a,c]anthracene.

A new synthesis of dibenzo[a,c]anthracene (4) is described that features the generation, from tetrabromo-bis-triflate 1 and phenyllithium, of a 1,3,6-naphthotriyne (2) synthetic equivalent that is trapped with 3 equiv of furan to form Diels-Alder tris-adduct 3. A subsequent two-step deoxygenation of 3 represents the first synthesis of dibenz[a,c]anthracene (4) that involves a tandem aryne Diels-Alder cycloaddition-deoxygenation strategy.

Tonantzitlolone cytotoxicity toward renal cancer cells is PKCθ- and HSF1-dependent.

Elucidating the targets and mechanism of action of natural products is strategically important prior to drug development and assessment of potential clinical applications. In this report, we elucidated the main targets and mechanism of action of the natural product tonantzitlolone (TZL) in clear cell renal cell carcinoma (CCRCC). We identified TZL as a dual PKCα and PKCθ activator in vitro, although in CCRCC cells its activity was mostly PKCθ-dependent. Through activation of PKCθ, TZL induced an insulin resistant phenotype by inhibiting IRS1 and the PI3K/Akt pathway. Simultaneously, TZL activated the heat shock factor 1 (HSF1) transcription factor driving glucose dependency. Thus, similar to the selective PKCθ activator englerin A, TZL induces a metabolic catastrophe in CCRCC, starving cells of glucose while simultaneously increasing their glycolytic dependency.

Targeting ABL1-mediated oxidative stress adaptation in fumarate hydratase-deficient cancer.

Patients with germline fumarate hydratase (FH) mutation are predisposed to develop aggressive kidney cancer with few treatment options and poor therapeutic outcomes. Activity of the proto-oncogene ABL1 is upregulated in FH-deficient kidney tumors and drives a metabolic and survival signaling network necessary to cope with impaired mitochondrial function and abnormal accumulation of intracellular fumarate. Excess fumarate indirectly stimulates ABL1 activity, while restoration of wild-type FH abrogates both ABL1 activation and the cytotoxicity caused by ABL1 inhibition or knockdown. ABL1 upregulates aerobic glycolysis via the mTOR/HIF1α pathway and neutralizes fumarate-induced proteotoxic stress by promoting nuclear localization of the antioxidant response transcription factor NRF2. Our findings identify ABL1 as a pharmacologically tractable therapeutic target in glycolytically dependent, oxidatively stressed tumors.