Monocyte phenotypes: when local education counts

Monocytes are a heterogeneous population of phagocytic cells that are generated in the bone marrow and released into the bloodstream. There are two main monocyte subsets in mice: “inflammatory” monocytes that are Ly6Chi CCR2hi CX3 CR1low and “alternative” or “patrolling” monocytes that are Ly6Clow CCR2low CX3 CR1hi. The process of monocyte recruitment and differentiation is still a matter of controversy. In this issue, Dal-Secco et al. report in situ monocyte reprogramming in the liver, from proinflammatory CCR2hi CX3 CR1low cells into reparative CCR2low CX3 CR1hi cells and show, for the first time, that this occurs at the site of injury.Fil: Quintar, Amado Alfredo. la Jolla Institute For Allergy And Immunology; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Hedrick, Catherine C. . la Jolla Institute For Allergy And Immunology; Estados UnidosFil: Ley, Klaus. la Jolla Institute For Allergy And Immunology; Estados Unido

CyTOF mass cytometry reveals phenotypically distinct human blood neutrophil populations differentially correlated with melanoma stage

BACKGROUND: Understanding neutrophil heterogeneity and its relationship to disease progression has become a recent focus of cancer research. Indeed, several studies have identified neutrophil subpopulations associated with protumoral or antitumoral functions. However, this work has been hindered by a lack of widely accepted markers with which to define neutrophil subpopulations. METHODS: To identify markers of neutrophil heterogeneity in cancer, we used single-cell cytometry by time-of-flight (CyTOF) coupled with high-dimensional analysis on blood samples from treatment-naïve patients with melanoma. RESULTS: Our efforts allowed us to identify seven blood neutrophil clusters, including two previously identified individual populations. Interrogation of these neutrophil subpopulations revealed a positive trend between specific clusters and disease stage. Finally, we recapitulated these seven blood neutrophil populations via flow cytometry and found that they exhibited diverse capacities for phagocytosis and reactive oxygen species production in vitro. CONCLUSIONS: Our data provide a refined consensus on neutrophil heterogeneity markers, enabling a prospective functional evaluation in patients with solid tumors.</p

Human Lipoxygenase Pathway Gene Variation and Association with Markers of Subclinical Atherosclerosis in the Diabetes Heart Study

Aims. Genes of the 5-lipoxygenase pathway are compelling candidates for atherosclerosis. We hypothesize that polymorphisms in ALOX12, ALOX15, ALOX5, and ALOX5AP genes are associated with subclinical atherosclerosis in multiple vascular beds. Methods. Families with two or more siblings with type 2 diabetes and their nondiabetic siblings were studied as part of the Diabetes Heart Study (DHS). European American diabetic (n = 828) and nondiabetic (n = 170) siblings were genotyped for SNPs in the ALOX12, ALOX15, ALOX5, and ALOX5AP genes. Subclinical measures of atherosclerosis (IMT, coronary (CorCP), carotid (CarCP) and aortic (AorCP) calcified plaque) were obtained. Results. Associations were observed between ALOX12 with CorCP, ALOX5 with CorCP, AorCP, and IMT, and ALOX5AP with CorCP and CarCP, independent of known epidemiologic risk factors. Further, lipoxygenase pathway SNPs that were associated with measures of atherosclerosis were associated with markers of inflammation (CRP, ICAM-1) and calcification (MGP). Conclusions. Polymorphisms within ALOX12, ALOX5, and ALOX5AP are genetically associated with subclinical atherosclerosis and with biomarkers of disease in families with type 2 diabetes. These results suggest that variants in lipoxygenase pathway genes may have pleiotropic effects on multiple components that determine risk of cardiovascular disease

Oxidized phospholipids are proinflammatory and proatherogenic in hypercholesterolaemic mice.

Oxidized phospholipids (OxPL) are ubiquitous, are formed in many inflammatory tissues, including atherosclerotic lesions, and frequently mediate proinflammatory changes 1 . Because OxPL are mostly the products of non-enzymatic lipid peroxidation, mechanisms to specifically neutralize them are unavailable and their roles in vivo are largely unknown. We previously cloned the IgM natural antibody E06, which binds to the phosphocholine headgroup of OxPL, and blocks the uptake of oxidized low-density lipoprotein (OxLDL) by macrophages and inhibits the proinflammatory properties of OxPL2-4. Here, to determine the role of OxPL in vivo in the context of atherogenesis, we generated transgenic mice in the Ldlr-/- background that expressed a single-chain variable fragment of E06 (E06-scFv) using the Apoe promoter. E06-scFv was secreted into the plasma from the liver and macrophages, and achieved sufficient plasma levels to inhibit in vivo macrophage uptake of OxLDL and to prevent OxPL-induced inflammatory signalling. Compared to Ldlr-/- mice, Ldlr -/- E06-scFv mice had 57-28% less atherosclerosis after 4, 7 and even 12 months of 1% high-cholesterol diet. Echocardiographic and histologic evaluation of the aortic valves demonstrated that E06-scFv ameliorated the development of aortic valve gradients and decreased aortic valve calcification. Both cholesterol accumulation and in vivo uptake of OxLDL were decreased in peritoneal macrophages, and both peritoneal and aortic macrophages had a decreased inflammatory phenotype. Serum amyloid A was decreased by 32%, indicating decreased systemic inflammation, and hepatic steatosis and inflammation were also decreased. Finally, the E06-scFv prolonged life as measured over 15 months. Because the E06-scFv lacks the functional effects of an intact antibody other than the ability to bind OxPL and inhibit OxLDL uptake in macrophages, these data support a major proatherogenic role of OxLDL and demonstrate that OxPL are proinflammatory and proatherogenic, which E06 counteracts in vivo. These studies suggest that therapies inactivating OxPL may be beneficial for reducing generalized inflammation, including the progression of atherosclerosis, aortic stenosis and hepatic steatosis

The transcription factor NR4A1 is essential for the development of a novel macrophage subset in the thymus

Tissue macrophages function to maintain homeostasis and regulate immune responses. While tissue macrophages derive from one of a small number of progenitor programs, the transcriptional requirements for site-specific macrophage subset development are more complex. We have identified a new tissue macrophage subset in the thymus and have discovered that its development is dependent on transcription factor NR4A1. Functionally, we find that NR4A1-dependent macrophages are critically important for clearance of apoptotic thymocytes. These macrophages are largely reduced or absent in mice lacking NR4A1, and Nr4a1-deficient mice have impaired thymocyte engulfment and clearance. Thus, NR4A1 functions as a master transcription factor for the development of this novel thymus-specific macrophage subset

CD33 Expression on Peripheral Blood Monocytes Predicts Efficacy of Anti-PD-1 Immunotherapy Against Non-Small Cell Lung Cancer

Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related deaths globally. Immune checkpoint blockade (ICB) has transformed cancer medicine, with anti-programmed cell death protein 1 (anti-PD-1) therapy now well-utilized for treating NSCLC. Still, not all patients with NSCLC respond positively to anti-PD-1 therapy, and some patients acquire resistance to treatment. There remains an urgent need to find markers predictive of anti-PD-1 responsiveness. To this end, we performed mass cytometry on peripheral blood mononuclear cells from 26 patients with NSCLC during anti-PD-1 treatment. Patients who responded to anti-PD-1 ICB displayed significantly higher levels of antigen-presenting myeloid cells, including CD9+ nonclassical monocytes, and CD33hi classical monocytes. Using matched pre-post treatment samples, we found that the baseline pre-treatment frequencies of CD33hi monocytes predicted patient responsiveness to anti-PD-1 therapy. Moreover, some of these classical and nonclassical monocyte subsets were associated with reduced immunosuppression by T regulatory (CD4+FOXP3+CD25+) cells in the same patients. Our use of machine learning corroborated the association of specific monocyte markers with responsiveness to ICB. Our work provides a high-dimensional profile of monocytes in NSCLC and links CD33 expression on monocytes with anti-PD-1 effectiveness in patients with NSCLC

Identification of an Early Unipotent Neutrophil Progenitor with Pro-tumoral Activity in Mouse and Human Bone Marrow

Neutrophils are short-lived cells that play important roles in both health and disease. Neutrophils and monocytes originate from the granulocyte monocyte progenitor (GMP) in bone marrow; however, unipotent neutrophil progenitors are not well defined. Here, we use cytometry by time of flight (CyTOF) and single-cell RNA sequencing (scRNA-seq) methodologies to identify a committed unipotent early-stage neutrophil progenitor (NeP) in adult mouse bone marrow. Importantly, we found a similar unipotent NeP (hNeP) in human bone marrow. Both NeP and hNeP generate only neutrophils. NeP and hNeP both significantly increase tumor growth when transferred into murine cancer models, including a humanized mouse model. hNeP are present in the blood of treatment-naive melanoma patients but not of healthy subjects. hNeP can be readily identified by flow cytometry and could be used as a biomarker for early cancer discovery. Understanding the biology of hNeP should allow the development of new therapeutic targets for neutrophil-related diseases, including cancer

Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9(+) monocytes associated with COVID-19 hospitalization

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression

Human CD79b⁺ neutrophils in the blood are associated with early-stage melanoma.

PurposeDue to their abundance in the blood, low RNA content, and short lifespan, neutrophils have been classically considered to be one homogenous pool. However, recent work has found that mature neutrophils and neutrophil progenitors are composed of unique subsets exhibiting context-dependent functions. In this study, we ask if neutrophil heterogeneity is associated with melanoma incidence and/or disease stage.Experimental designUsing mass cytometry, we profiled melanoma patient blood for unique cell surface markers among neutrophils. Markers were tested for their predictiveness using flow cytometry data and random forest machine learning.ResultsWe identified CD79b+ neutrophils (CD3-CD56-CD19-Siglec8-CD203c-CD86LoCD66b+CD79b+) that are normally restricted to the bone marrow in healthy humans but appear in the blood of subjects with early-stage melanoma. Further, we found CD79b+ neutrophils present in tumors of subjects with head and neck cancer. AI-mediated machine learning analysis of neutrophils from subjects with melanoma confirmed that CD79b expression among peripheral blood neutrophils is highly important in identifying melanoma incidence. We noted that CD79b+ neutrophils possessed a neutrophilic appearance but have transcriptional and surface-marker phenotypes reminiscent of B cells. Compared to remaining blood neutrophils, CD79b+ neutrophils are primed for NETosis, express higher levels of antigen presentation-related proteins, and have an increased capacity for phagocytosis.ConclusionOur work suggests that CD79b+ neutrophils are associated with early-stage melanoma

Pelvic trauma : WSES classification and guidelines

Complex pelvic injuries are among the most dangerous and deadly trauma related lesions. Different classification systems exist, some are based on the mechanism of injury, some on anatomic patterns and some are focusing on the resulting instability requiring operative fixation. The optimal treatment strategy, however, should keep into consideration the hemodynamic status, the anatomic impairment of pelvic ring function and the associated injuries. The management of pelvic trauma patients aims definitively to restore the homeostasis and the normal physiopathology associated to the mechanical stability of the pelvic ring. Thus the management of pelvic trauma must be multidisciplinary and should be ultimately based on the physiology of the patient and the anatomy of the injury. This paper presents the World Society of Emergency Surgery (WSES) classification of pelvic trauma and the management Guidelines.Peer reviewe