Characterization of a New Immunosuppressive and Antimicrobial Peptide, DRS-DA2, Isolated from the Mexican Frog, Pachymedusa dacnicolor.

Inflammatory and antimicrobial diseases constitute a major burden for society, and fighting them is a WHO strategic priority. Most of the treatments available to fight inflammatory diseases are anti-inflammatory drugs, such as corticosteroids or immunomodulators that lack cellular specificity and lead to numerous side effects. In addition to suppressing undesired inflammation and reducing disease progression, these drugs lessen the immune system protective functions. Furthermore, treating infectious diseases is more and more challenging due to the rise of microbial resistance to antimicrobial drugs. Thus, controlling the inflammatory process locally without compromising the ability to combat infections is an essential feature in the treatment of inflammatory diseases. We isolated three forms (DRS-DA2N, DRS-DA2NE, and DRS-DA2NEQ) of the same peptide, DRS-DA2, which belongs to the dermaseptin family, from the Mexican tree frog Pachymedusa dacnicolor. Interestingly, DRS-DA2N and DRS-DA2NEQ exhibit a dual activity by inducing the death of leukocytes as well as that of Gram-negative and Gram-positive bacteria, including multiresistant strains, without affecting other cells such as epithelial cells or erythrocytes. We showed that the death of both immune cells and bacteria is induced rapidly by DRS-DA2 and that the membrane is permeabilized, leading to the loss of membrane integrity. We also validated the capacity of DRS-DA2 to regulate the pool of inflammatory cells in vivo in a mouse model of noninfectious peritonitis. After the induction of peritonitis, a local injection of DRS-DA2N could decrease the number of inflammatory cells locally in the peritoneal cavity without inducing a systemic effect, as no changes in the number of inflammatory cells could be detected in blood or in the bone marrow. Collectively, these data suggest that this peptide could be a promising tool in the treatment of inflammatory diseases, such as inflammatory skin diseases, as it could reduce the number of inflammatory cells locally without suppressing the ability to combat infections.

Chemokine Receptor 2 Is A Theranostic Biomarker for Abdominal Aortic Aneurysms.

Abdominal aortic aneurysm (AAA) is a degenerative vascular disease impacting aging populations with a high mortality upon rupture. There are no effective medical therapies to prevent AAA expansion and rupture. We previously demonstrated the role of the monocyte chemoattractant protein-1 (MCP-1) / C-C chemokine receptor type 2 (CCR2) axis in rodent AAA pathogenesis via positron emission tomography/computed tomography (PET/CT) using CCR2 targeted radiotracer 64 Cu-DOTA-ECL1i. We have since translated this radiotracer into patients with AAA. CCR2 PET showed intense radiotracer uptake along the AAA wall in patients while little signal was observed in healthy volunteers. AAA tissues collected from individuals scanned with 64 Cu-DOTA-ECL1i and underwent open-repair later demonstrated more abundant CCR2+ cells compared to non-diseased aortas. We then used a CCR2 inhibitor (CCR2i) as targeted therapy in our established male and female rat AAA rupture models. We observed that CCR2i completely prevented AAA rupture in male rats and significantly decreased rupture rate in female AAA rats. PET/CT revealed substantial reduction of 64 Cu-DOTA-ECL1i uptake following CCR2i treatment in both rat models. Characterization of AAA tissues demonstrated decreased expression of CCR2+ cells and improved histopathological features. Taken together, our results indicate the potential of CCR2 as a theranostic biomarker for AAA management.

Memory CD4+ T-Cell Lymphocytic Angiopathy in Fatal Forms of COVID-19 Pulmonary Infection.

The immunopathological pulmonary mechanisms leading to Coronavirus Disease (COVID-19)-related death in adults remain poorly understood. Bronchoalveolar lavage (BAL) and peripheral blood sampling were performed in 74 steroid and non-steroid-treated intensive care unit (ICU) patients (23-75 years; 44 survivors). Peripheral effector SARS-CoV-2-specific T cells were detected in 34/58 cases, mainly directed against the S1 portion of the spike protein. The BAL lymphocytosis consisted of T cells, while the mean CD4/CD8 ratio was 1.80 in non-steroid- treated patients and 1.14 in steroid-treated patients. Moreover, strong BAL SARS-CoV-2 specific T-cell responses were detected in 4/4 surviving and 3/3 non-surviving patients. Serum IFN-γ and IL-6 levels were decreased in steroid-treated patients when compared to non-steroid treated patients. In the lung samples from 3 (1 non-ICU and 2 ICU) additional deceased cases, a lymphocytic memory CD4 T-cell angiopathy colocalizing with SARS-CoV-2 was also observed. Taken together, these data show that disease severity occurs despite strong antiviral CD4 T cell-specific responses migrating to the lung, which could suggest a pathogenic role for perivascular memory CD4 T cells upon fatal COVID-19 pneumonia.

Tumor-associated macrophage heterogeneity is driven by tissue territories in breast cancer.

Tissue-resident macrophages adapt to local signals within tissues to acquire specific functions. Neoplasia transforms the tissue, raising the question as to how the environmental perturbations contribute to tumor-associated macrophage (TAM) identity and functions. Combining single-cell RNA sequencing (scRNA-seq) with spatial localization of distinct TAM subsets by imaging, we discover that TAM transcriptomic programs follow two main differentiation paths according to their localization in the stroma or in the neoplastic epithelium of the mammary duct. Furthermore, this diversity is exclusively detected in a spontaneous tumor model and tracks the different tissue territories as well as the type of tumor lesion. These TAM subsets harbor distinct capacity to activate CD8+ T cells and phagocyte tumor cells, supporting that specific tumor regions, rather than defined activation states, are the major drivers of TAM plasticity and heterogeneity. The distinctions created here provide a framework to design cancer treatment targeting specific TAM niches.

Two New Neutrophil Subsets Define a Discriminating Sepsis Signature.

Rationale: Sepsis is the leading cause of death in adult ICUs. At present, sepsis diagnosis relies on nonspecific clinical features. It could transform clinical care to have immune-cell biomarkers that could predict sepsis diagnosis and guide treatment. For decades, neutrophil phenotypes have been studied in sepsis, but a diagnostic cell subset has yet to be identified. Objectives: To identify an early, specific immune signature of sepsis severity that does not overlap with other inflammatory biomarkers and that distinguishes patients with sepsis from those with noninfectious inflammatory syndrome. Methods: Mass cytometry combined with computational high-dimensional data analysis was used to measure 42 markers on whole-blood immune cells from patients with sepsis and control subjects and to automatically and comprehensively characterize circulating immune cells, which enables identification of novel, disease-specific cellular signatures. Measurements and Main Results: Unsupervised analysis of high-dimensional mass cytometry data characterized previously unappreciated heterogeneity within the CD64+ immature neutrophils and revealed two new subsets distinguished by CD123 and PD-L1 (programmed death ligand 1) expression. These immature neutrophils exhibited diminished activation and phagocytosis functions. The proportion of CD123-expressing neutrophils correlated with clinical severity. Conclusions: This study showed that these two new neutrophil subsets were specific to sepsis and detectable through routine flow cytometry by using seven markers. The demonstration here that a simple blood test distinguishes sepsis from other inflammatory conditions represents a key biological milestone that can be immediately translated into improvements in patient care.

LOX-1-Expressing Immature Neutrophils Identify Critically-Ill COVID-19 Patients at Risk of Thrombotic Complications.

Background: Lymphopenia and the neutrophil/lymphocyte ratio may have prognostic value in COVID-19 severity. Objective: We investigated neutrophil subsets and functions in blood and bronchoalveolar lavage (BAL) of COVID-19 patients on the basis of patients' clinical characteristics. Methods: We used a multiparametric cytometry profiling based to mature and immature neutrophil markers in 146 critical or severe COVID-19 patients. Results: The Discovery study (38 patients, first pandemic wave) showed that 80% of Intensive Care Unit (ICU) patients develop strong myelemia with CD10-CD64+ immature neutrophils (ImNs). Cellular profiling revealed three distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the interleukin-3 receptor alpha (CD123), or programmed death-ligand 1 (PD-L1) overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1- or CD123-expressing ImNs is positively correlated with clinical severity, cytokine storm (IL-1ß, IL-6, IL-8, TNFα), acute respiratory distress syndrome (ARDS), and thrombosis. BALs of patients with ARDS were highly enriched in LOX-1-expressing ImN subsets and in antimicrobial neutrophil factors. A validation study (118 patients, second pandemic wave) confirmed and strengthened the association of the proportion of ImN subsets with disease severity, invasive ventilation, and death. Only high proportions of LOX-1-expressing ImNs remained strongly associated with a high risk of severe thrombosis independently of the plasma antimicrobial neutrophil factors, suggesting an independent association of ImN markers with their functions. Conclusion: LOX-1-expressing ImNs may help identifying COVID-19 patients at high risk of severity and thrombosis complications.

Elevated Neopterin Levels Predict Fatal Outcome in SARS-CoV-2-Infected Patients.

Highlights: Innate immune activation during Covid-19 infection is associated with pernicious clinical outcome. Background: Coronavirus disease 2019 (Covid-19) is a worldwide threat that has already caused more than 3 000 000 deaths. It is characterized by different patterns of disease evolution depending on host factors among which old-age and pre-existing comorbidities play a detrimental role. Previous coronavirus epidemics, notably SARS-CoV, were associated with increased serum neopterin levels, which can be interpreted as a sign of acute innate immunity in response to viral infection. Here we hypothesize that neopterin may serve as a biomarker of SARS-CoV-2 viral infection and Covid-19 disease severity. Methods: We measured neopterin blood levels by ELISA. Seric concentration was quantified from 256 healthy donors and 374 Covid-19 patients at hospital admission. Enrolled Covid-19 patients were all symptomatic and displayed a large spectrum of comorbidities. Patients were followed until disease resolution or death. Results: Severe and critically ill SARS-CoV-2 infected patients were characterized by a profound exacerbation of immune activation characterized by elevated neopterin blood levels. Systemic neopterin levels above 19nM stratified healthy individuals from Covid-19 patients with 87% specificity and 100% sensitivity. Moreover, systemic neopterin levels above 53nM differentiated non-survivors from survivors with 64% specificity and 100% sensitivity. Conclusion: We propose that neopterin concentration measured at arrival to hospital is a hallmark of severe Covid-19 and identifies a high-risk population of pernicious clinical outcome with a need for special medical care.

CD8+PD-L1+CXCR3+ polyfunctional T cell abundances are associated with survival in critical SARS-CoV-2-infected patients.

The importance of the adaptive T cell response in the control and resolution of viral infection has been well established. However, the nature of T cell-mediated viral control mechanisms in life-threatening stages of COVID-19 has yet to be determined. The aim of the present study was to determine the function and phenotype of T cell populations associated with survival or death of patients with COVID-19 in intensive care as a result of phenotypic and functional profiling by mass cytometry. Increased frequencies of circulating, polyfunctional CD4+CXCR5+HLA-DR+ stem cell memory T cells (Tscms) and decreased proportions of granzyme B-expressing and perforin-expressing effector memory T cells were detected in recovered and deceased patients, respectively. The higher abundance of polyfunctional PD-L1+CXCR3+CD8+ effector T cells (Teffs), CXCR5+HLA-DR+ Tscms, and anti-nucleocapsid (anti-NC) cytokine-producing T cells permitted us to differentiate between recovered and deceased patients. The results from a principal component analysis show an imbalance in the T cell compartment that allowed for the separation of recovered and deceased patients. The paucity of circulating PD-L1+CXCR3+CD8+ Teffs and NC-specific CD8+ T cells accurately forecasts fatal disease outcome. This study provides insight into the nature of the T cell populations involved in the control of COVID-19 and therefore might impact T cell-based vaccine designs for this infectious disease.

Distinct cytokine profiles associated with COVID-19 severity and mortality.

BACKGROUND: Markedly elevated levels of proinflammatory cytokines and defective type-I interferon responses were reported in patients with coronavirus disease 2019 (COVID-19). OBJECTIVE: We sought to determine whether particular cytokine profiles are associated with COVID-19 severity and mortality. METHODS: Cytokine concentrations and severe acute respiratory syndrome coronavirus 2 antigen were measured at hospital admission in serum of symptomatic patients with COVID-19 (N = 115), classified at hospitalization into 3 respiratory severity groups: no need for mechanical ventilatory support (No-MVS), intermediate severity requiring mechanical ventilatory support (MVS), and critical severity requiring extracorporeal membrane oxygenation (ECMO). Principal-component analysis was used to characterize cytokine profiles associated with severity and mortality. The results were thereafter confirmed in an independent validation cohort (N = 86). RESULTS: At time of hospitalization, ECMO patients presented a dominant proinflammatory response with elevated levels of TNF-α, IL-6, IL-8, and IL-10. In contrast, an elevated type-I interferon response involving IFN-α and IFN-ß was characteristic of No-MVS patients, whereas MVS patients exhibited both profiles. Mortality at 1 month was associated with higher levels of proinflammatory cytokines in ECMO patients, higher levels of type-I interferons in No-MVS patients, and their combination in MVS patients, resulting in a combined mortality prediction accuracy of 88.5% (risk ratio, 24.3; P < .0001). Severe acute respiratory syndrome coronavirus 2 antigen levels correlated with type-I interferon levels and were associated with mortality, but not with proinflammatory response or severity. CONCLUSIONS: Distinct cytokine profiles are observed in association with COVID-19 severity and are differentially predictive of mortality according to oxygen support modalities. These results warrant personalized treatment of COVID-19 patients based on cytokine profiling.

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

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-1ß 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).

Imaging resident and recruited macrophage contribution to Wallerian degeneration.

Wallerian degeneration (WD) is a process of autonomous distal degeneration of axons upon injury. Macrophages (MPs) of the peripheral nervous system (PNS) are the main cellular agent controlling this process. Some evidence suggests that resident PNS-MPs along with MPs of hematogenous origin may be involved, but whether these two subsets exert distinct functions is unknown. Combining MP-designed fluorescent reporter mice and coherent anti-Stokes Raman scattering (CARS) imaging of the sciatic nerve, we deciphered the spatiotemporal choreography of resident and recently recruited MPs after injury and unveiled distinct functions of these subsets, with recruited MPs being responsible for efficient myelin stripping and clearance and resident MPs being involved in axonal regrowth. This work provides clues to tackle selectively cellular processes involved in neurodegenerative diseases.

CX3CL1 homo-oligomerization drives cell-to-cell adherence.

During inflammatory response, blood leukocytes adhere to the endothelium. This process involves numerous adhesion molecules, including a transmembrane chemokine, CX3CL1, which behaves as a molecular cluster. How this cluster assembles and whether this association has a functional role remain unknown. The analysis of CX3CL1 clusters using native electrophoresis and single molecule fluorescence kinetics shows that CX3CL1 is a homo-oligomer of 3 to 7 monomers. Fluorescence recovery after photobleaching assays reveal that the CX3CL1-transmembrane domain peptide self-associates in both cellular and acellular lipid environments, while its random counterpart (i.e. peptide with the same residues in a different order) does not. This strongly indicates that CX3CL1 oligomerization is driven by its intrinsic properties. According to the molecular modeling, CX3CL1 does not associate in compact bundles but rather with monomers linearly assembled side by side. Finally, the CX3CL1 transmembrane peptide inhibits both the CX3CL1 oligomerization and the adhesive function, while its random counterpart does not. This demonstrates that CX3CL1 oligomerization is mandatory for its adhesive potency. Our results provide a new direction to control CX3CL1-dependent cellular adherence in key immune processes.

Revising CX3CR1 Expression on Murine Classical and Non-classical Monocytes.

In mice, monocytes (Mo) are conventionally described as CX3CR1low classical Mo (CMo) and CX3CR1high non-classical Mo (NCMo) based on the expression of EGFP in Cx3cr1+/EGFP mice and by analogy with human CX3CR1 expression. Although this terminology is widely used, it may not reflect the expression of CX3CR1 on Mo subsets. Using an unsupervised multiparametric analysis of blood Mo in steady state and after sterile peritonitis, we observed that CX3CR1 expression did not discriminate the CMo from the NCMo subsets. Our results highlight that despite being a reliable reporter to discriminate Mo subpopulations, EGFP level in Cx3cr1+/EGFP mice does not reflect CX3CR1 expression measured by a fluorescently-labeled CX3CL1 chemokine and a CX3CR1 specific antibody. In conclusion, authors should be cautious not to identify murine classical and non-classical Mo as CX3CR1low and CX3CR1high but rather use alternative markers such as the combination of Ly6C and CD43.

Sepsis Triggers a Late Expansion of Functionally Impaired Tissue-Vascular Inflammatory Monocytes During Clinical Recovery.

Sepsis is characterized by a systemic inflammation that can cause an immune dysfunction, for which the underlying mechanisms are unclear. We investigated the impact of cecal ligature and puncture (CLP)-mediated polymicrobial sepsis on monocyte (Mo) mobilization and functions. Our results show that CLP led to two consecutive phases of Mo deployment. The first one occurred within the first 3 days after the induction of the peritonitis, while the second phase was of a larger amplitude and extended up to a month after apparent clinical recovery. The latter was associated with the expansion of Mo in the tissue reservoirs (bone marrow and spleen), their release in the blood and their accumulation in the vasculature of peripheral non-lymphoid tissues. It occurred even after antibiotic treatment but relied on inflammatory-dependent pathways and inversely correlated with increased susceptibility and severity to a secondary infection. The intravascular lung Mo displayed limited activation capacity, impaired phagocytic functions and failed to transfer efficient protection against a secondary infection into monocytopenic CCR2-deficient mice. In conclusion, our work unveiled key dysfunctions of intravascular inflammatory Mo during the recovery phase of sepsis and provided new insights to improve patient protection against secondary infections.

Phagocytosis of Wnt inhibitor SFRP4 by late wound macrophages drives chronic Wnt activity for fibrotic skin healing.

Human and murine skin wounding commonly results in fibrotic scarring, but the murine wounding model wound-induced hair neogenesis (WIHN) can frequently result in a regenerative repair response. Here, we show in single-cell RNA sequencing comparisons of semi-regenerative and fibrotic WIHN wounds, increased expression of phagocytic/lysosomal genes in macrophages associated with predominance of fibrotic myofibroblasts in fibrotic wounds. Investigation revealed that macrophages in the late wound drive fibrosis by phagocytizing dermal Wnt inhibitor SFRP4 to establish persistent Wnt activity. In accordance, phagocytosis abrogation resulted in transient Wnt activity and a more regenerative healing. Phagocytosis of SFRP4 was integrin-mediated and dependent on the interaction of SFRP4 with the EDA splice variant of fibronectin. In the human skin condition hidradenitis suppurativa, phagocytosis of SFRP4 by macrophages correlated with fibrotic wound repair. These results reveal that macrophages can modulate a key signaling pathway via phagocytosis to alter the skin wound healing fate.

Ibrutinib induces multiple functional defects in the neutrophil response against Aspergillus fumigatus.

The Bruton tyrosine kinase inhibitor ibrutinib has become a leading therapy against chronic lymphoid leukemia. Recently, ibrutinib has been associated with the occurrence of invasive fungal infections, in particular invasive aspergillosis. The mechanisms underlying the increased susceptibility to fungal infections associated with exposure to ibrutinib are currently unknown. Innate immunity, in particular polymer-phonuclear neutrophils, represents the cornerstone of anti-Aspergillus immunity; however, the potential impact of ibrutinib on neutrophils has been little studied. Our study investigated the response to Aspergillus fumigatus and neutrophil function in patients with chronic lymphoid leukemia or lymphoma, who were undergoing ibrutinib therapy. We studied the consequences of ibrutinib exposure on the functions and anti-Aspergillus responses of neutrophils obtained from healthy donors and 63 blood samples collected at different time points from 32 patients receiving ibrutinib for lymphoid malignancies. We used both flow cytometry and video-microscopy approaches to analyze neutrophils' cell surface molecule expression, cytokine production, oxidative burst, chemotaxis and killing activity against Aspergillus Ibrutinib is associated, both in vitro and in patients under treatment, with multiple functional defects in neutrophils, including decreased production of reactive oxygen species, impairment of their capacity to engulf Aspergillus and inability to efficiently kill germinating conidia. Our results demonstrate that ibrutinib-exposed neutrophils develop significant functional defects that impair their response against Aspergillus fumigatus, providing a plausible explanation for the emergence of invasive aspergillosis in ibrutinib-treated patients.

Tracking Monocytes and Macrophages in Tumors With Live Imaging.

In most cancers, myeloid cells represent the major component of the immune microenvironment. Deciphering the impact of these cells on tumor growth and in response to various anti-tumor therapies is a key issue. Many studies have elucidated the role of tumor-associated monocytes and tumor-associated macrophages (TAM) in tumor development, angiogenesis, and therapeutic failure. In contrast, tumor dendritic cells (DC) are associated with tumor antigen uptake and T-cell priming. Myeloid subpopulations display differences in ontogeny, state of differentiation and distribution within the neoplastic tissue, making them difficult to study. The development of high-dimensional genomic and cytometric analyses has unveiled the large functional diversity of myeloid cells. Important fundamental insights on the biology of myeloid cells have also been provided by a boom in functional fluorescent imaging techniques, in particular for TAM. These approaches allow the tracking of cell behavior in native physiological environments, incorporating spatio-temporal dimensions in the study of their functional activity. Nevertheless, tracking myeloid cells within the TME remains a challenging process as many markers overlap between monocytes, macrophages, DC, and neutrophils. Therefore, perfect discrimination between myeloid subsets remains impossible to date. Herein we review the specific functions of myeloid cells in tumor development unveiled by image-based tracking, the limits of fluorescent reporters commonly used to accurately track specific myeloid cells, and novel combinations of myeloid-associated fluorescent reporters that better discriminate the relative contributions of these cells to tumor biology according to their origin and tissue localization.

CCR2-Dependent Recruitment of Tregs and Monocytes Following Radiotherapy Is Associated with TNFα-Mediated Resistance.

Radiotherapy (RT) represents one of the main anticancer approaches for the treatment of solid tumors. Beyond the expected direct effects of RT on tumor cells, evidence supporting the importance of an immune response to RT is growing. The balance between RT-mediated immunogenic and tolerogenic activity is ill-defined and deserves more attention. Herein, a murine model of head and neck squamous cell carcinoma was used to demonstrate that RT upregulated CCL2 chemokine production in tumor cells, leading to a CCR2-dependent accumulation of tumor necrosis factor alpha (TNFα)-producing monocytes and CCR2+ regulatory T cells (Treg). This corecruitment was associated with a TNFα-dependent activation of Tregs, dampening the efficacy of RT. Our results highlight an unexpected cross-talk between innate and adaptive immune system components and indicate CCL2/CCR2 and TNFα as potential clinical candidates to counterbalance the radioprotective action of monocyte-derived cells and Tregs, paving the way for potent combined radioimmunotherapies.

Molecular Imaging Visualizes Recruitment of Inflammatory Monocytes and Macrophages to the Injured Heart.

RATIONALE: Paradigm shifting studies have revealed that the heart contains functionally diverse populations of macrophages derived from distinct embryonic and adult hematopoietic progenitors. Under steady-state conditions, the heart is largely populated by CCR2- (C-C chemokine receptor type 2) macrophages of embryonic descent. After tissue injury, a dramatic shift in macrophage composition occurs whereby CCR2+ monocytes are recruited to the heart and differentiate into inflammatory CCR2+ macrophages that contribute to heart failure progression. Currently, there are no techniques to noninvasively detect CCR2+ monocyte recruitment into the heart and thus identify patients who may be candidates for immunomodulatory therapy. OBJECTIVE: To develop a noninvasive molecular imaging strategy with high sensitivity and specificity to visualize inflammatory monocyte and macrophage accumulation in the heart. METHODS AND RESULTS: We synthesized and tested the performance of a positron emission tomography radiotracer (68Ga-DOTA [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ECL1i [extracellular loop 1 inverso]) that allosterically binds to CCR2. In naive mice, the radiotracer was quickly cleared from the blood and displayed minimal retention in major organs. In contrast, biodistribution and positron emission tomography demonstrated strong myocardial tracer uptake in 2 models of cardiac injury (diphtheria toxin induced cardiomyocyte ablation and reperfused myocardial infarction). 68Ga-DOTA-ECL1i signal localized to sites of tissue injury and was independent of blood pool activity as assessed by quantitative positron emission tomography and ex vivo autoradiography. 68Ga-DOTA-ECL1i uptake was associated with CCR2+ monocyte and CCR2+ macrophage infiltration into the heart and was abrogated in CCR2-/- mice, demonstrating target specificity. Autoradiography demonstrated that 68Ga-DOTA-ECL1i specifically binds human heart failure specimens and with signal intensity associated with CCR2+ macrophage abundance. CONCLUSIONS: These findings demonstrate the sensitivity and specificity of 68Ga-DOTA-ECL1i in the mouse heart and highlight the translational potential of this agent to noninvasively visualize CCR2+ monocyte recruitment and inflammatory macrophage accumulation in patients.