Mutation of the Transcriptional Regulator YtoI Rescues Listeria monocytogenes Mutants Deficient in the Essential Shared Metabolite 1,4-Dihydroxy-2-Naphthoate (DHNA).

Listeria monocytogenes, a Gram-positive, facultative intracellular pathogen, survives and replicates in the cytosol of host cells. Synthesis of 1,4-dihydroxy-2-naphthoate (DHNA), an intermediate of menaquinone biosynthesis, is essential for cytosolic survival of L. monocytogenes independent from its role in respiration. Here, we demonstrate that DHNA is essential for virulence in a murine model of listeriosis due to both respiration-dependent and -independent functions. In addition, DHNA can be both secreted and utilized as an extracellular shared metabolite to promote cytosolic survival inside host macrophages. To understand the role(s) of DHNA in L. monocytogenes intracellular survival and virulence, we isolated DHNA-deficient (ΔmenD strain) suppressor mutants that formed plaques in monolayers of fibroblasts. Five ΔmenD suppressor (mds) mutants additionally rescued at least 50% of the cytosolic survival defect of the parent ΔmenD mutant. Whole-genome sequencing revealed that four of the five suppressor mutants had independent missense mutations in a putative transcriptional regulator, ytoI (lmo1576). Clean deletion and complementation in trans confirmed that loss of ytoI could restore plaquing and cytosolic survival of DHNA-deficient L. monocytogenes RNA-seq transcriptome analysis revealed five genes (lmo0944, lmo1575, lmo1577, lmo2005, and lmo2006) expressed at a higher level in the ΔytoI strain than in the wild-type strain, whereas two genes (lmo1917 and lmo2103) demonstrated lower expression in the ΔytoI mutant. Intriguingly, the majority of these genes are involved in controlling pyruvate flux. Metabolic analysis confirmed that acetoin, acetate, and lactate flux were altered in a ΔytoI mutant, suggesting a critical role for regulating these metabolic programs. In conclusion, we have demonstrated that, similar to findings in select other bacteria, DHNA can act as a shared resource, and it is essential for cytosolic survival and virulence of L. monocytogenes Furthermore, we have identified a novel transcriptional regulator in L. monocytogenes and determined that its metabolic regulation is implicated in cytosolic survival of L. monocytogenes.

Development of a CCR2 targeted 18F-labeled radiotracer for atherosclerosis imaging with PET.

Atherosclerosis is a chronic inflammatory disease and the leading cause of morbidity and mortality worldwide. CC motif chemokine ligand 2 and its corresponding cognate receptor 2 (CCL2/CCR2) signaling has been implicated in regulating monocyte recruitment and macrophage polarization during inflammatory responses that plays a pivotal role in atherosclerosis initiation and progression. In this study, we report the design and synthesis of a novel 18F radiolabeled small molecule radiotracer for CCR2-targeted positron emission tomography (PET) imaging in atherosclerosis. The binding affinity of this radiotracer to CCR2 was evaluated via in vitro binding assay using CCR2+ membrane and cells. Ex vivo biodistribution was carried out in wild type mice to assess radiotracer pharmacokinetics. CCR2 targeted PET imaging of plaques was performed in two murine atherosclerotic models. The sensitive detection of atherosclerotic lesions highlighted the potential of this radiotracer for CCR2 targeted PET and warranted further optimization.

Development of a CD163-Targeted PET Radiotracer That Images Resident Macrophages in Atherosclerosis.

Tissue-resident macrophages are complementary to proinflammatory macrophages to promote the progression of atherosclerosis. The noninvasive detection of their presence and dynamic variation will be important to the understanding of their role in the pathogenesis of atherosclerosis. The goal of this study was to develop a targeted PET radiotracer for imaging CD163-positive (CD163+) macrophages in multiple mouse atherosclerosis models and assess the potential of CD163 as a biomarker for atherosclerosis in humans. Methods: CD163-binding peptide was identified using phage display and conjugated with a NODAGA chelator for 64Cu radiolabeling ([64Cu]Cu-ICT-01). CD163-overexpressing U87 cells were used to measure the binding affinity of [64Cu]Cu-ICT-01. Biodistribution studies were performed on wild-type C57BL/6 mice at multiple time points after tail vein injection. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages upregulated on the surface of atherosclerotic plaques were assessed in multiple mouse atherosclerosis models. Immunostaining, flow cytometry, and single-cell RNA sequencing were performed to characterize the expression of CD163 on tissue-resident macrophages. Human carotid atherosclerotic plaques were used to measure the expression of CD163+ resident macrophages and test the binding specificity of [64Cu]Cu-ICT-01. Results: [64Cu]Cu-ICT-01 showed high binding affinity to U87 cells. The biodistribution study showed rapid blood and renal clearance with low retention in all major organs at 1, 2, and 4 h after injection. In an ApoE-/- mouse model, [64Cu]Cu-ICT-01 demonstrated sensitive and specific detection of CD163+ macrophages and capability for tracking the progression of atherosclerotic lesions; these findings were further confirmed in Ldlr-/- and PCSK9 mouse models. Immunostaining showed elevated expression of CD163+ macrophages across the plaques. Flow cytometry and single-cell RNA sequencing confirmed the specific expression of CD163 on tissue-resident macrophages. Human tissue characterization demonstrated high expression of CD163+ macrophages on atherosclerotic lesions, and ex vivo autoradiography revealed specific binding of [64Cu]Cu-ICT-01 to human CD163. Conclusion: This work reported the development of a PET radiotracer binding CD163+ macrophages. The elevated expression of CD163+ resident macrophages on human plaques indicated the potential of CD163 as a biomarker for vulnerable plaques. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages warrant further investigation in translational settings.

Chemokine Receptor 2 Targeted PET/CT Imaging Distant Metastases in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and treatment-refractory malignancies. The lack of an effective screening tool results in the majority of patients being diagnosed at late stages, which underscores the urgent need to develop more sensitive and specific imaging modalities, particularly in detecting occult metastases, to aid clinical decision-making. The tumor microenvironment of PDAC is heavily infiltrated with myeloid-derived suppressor cells (MDSCs) that express C-C chemokine receptor type 2 (CCR2). These CCR2-expressing MDSCs accumulate at a very early stage of metastasis and greatly outnumber PDAC cells, making CCR2 a promising target for detecting early, small metastatic lesions that have scant PDAC cells. Herein, we evaluated a CCR2 targeting PET tracer (68Ga-DOTA-ECL1i) for PET imaging on PDAC metastasis in two mouse models. Positron emission tomography/computed tomography (PET/CT) imaging of 68Ga-DOTA-ECL1i was performed in a hemisplenic injection metastasis model (KI) and a genetically engineered orthotopic PDAC model (KPC), which were compared with 18F-FDG PET concurrently. Autoradiography, hematoxylin and eosin (H&E), and CCR2 immunohistochemical staining were performed to characterize the metastatic lesions. PET/CT images visualized the PDAC metastases in the liver/lung of KI mice and in the liver of KPC mice. Quantitative uptake analysis revealed increased metastasis uptake during disease progression in both models. In comparison, 18F-FDG PET failed to detect any metastases during the time course studies. H&E staining showed metastases in the liver and lung of KI mice, within which immunostaining clearly demonstrated the overexpression of CCR2 as well as CCR2+ cell infiltration into the normal liver. H&E staining, CCR2 staining, and autoradiography also confirmed the expression of CCR2 and the uptake of 68Ga-DOTA-ECL1i in the metastatic foci in KPC mice. Using our novel CCR2 targeted radiotracer 68Ga-DOTA-ECL1i and PET/CT, we demonstrated the sensitive and specific detection of CCR2 in the early PDAC metastases in two mouse models, indicating its potential in future clinical translation.

Retinal tissue and microvasculature loss in COVID-19 infection.

This cross-sectional study aimed to investigate the hypothesis that permanent capillary damage may underlie the long-term COVID-19 sequela by quantifying the retinal vessel integrity. Participants were divided into three subgroups; Normal controls who had not been affected by COVID-19, mild COVID-19 cases who received out-patient care, and severe COVID-19 cases requiring intensive care unit (ICU) admission and respiratory support. Patients with systemic conditions that may affect the retinal vasculature before the diagnosis of COVID-19 infection were excluded. Participants underwent comprehensive ophthalmologic examination and retinal imaging obtained from Spectral-Domain Optical Coherence Tomography (SD-OCT), and vessel density using OCT Angiography. Sixty-one eyes from 31 individuals were studied. Retinal volume was significantly decreased in the outer 3 mm of the macula in the severe COVID-19 group (p = 0.02). Total retinal vessel density was significantly lower in the severe COVID-19 group compared to the normal and mild COVID-19 groups (p = 0.004 and 0.0057, respectively). The intermediate and deep capillary plexuses in the severe COVID-19 group were significantly lower compared to other groups (p < 0.05). Retinal tissue and microvascular loss may be a biomarker of COVID-19 severity. Further monitoring of the retina in COVID-19-recovered patients may help further understand the COVID-19 sequela.