Defining the proteomic landscape of cultured macrophages and their polarization continuum.

Macrophages have previously been characterized based on phenotypical and functional differences into suggested simplified subtypes of MØ, M1, M2a and M2c. These macrophage subtypes can be generated in a well-established primary monocyte culture model that produces cells expressing accepted subtype surface markers. To determine how these subtypes retain functional similarities and better understand their formation, we generated all four subtypes from the same donors. Comparative whole-cell proteomics confirmed that four distinct macrophage subtypes could be induced from the same donor material, with > 50% of 5435 identified proteins being significantly altered in abundance between subtypes. Functional assessment highlighted that these distinct protein expression profiles are primed to enable specific cell functions, indicating that this shifting proteome is predictive of meaningful changes in cell characteristics. Importantly, the 2552 proteins remained consistent in abundance across all macrophage subtypes examined, demonstrating maintenance of a stable core proteome that likely enables swift polarity changes. We next explored the cross-polarization capabilities of preactivated M1 macrophages treated with dexamethasone. Importantly, these treated cells undergo a partial repolarization toward the M2c surface markers but still retain the M1 functional phenotype. Our investigation of polarized macrophage subtypes therefore provides evidence of a sliding scale of macrophage functionality, with these data sets providing a valuable benchmark resource for further studies of macrophage polarity, with relevance for cell therapy development and drug discovery.

Macrophage Reprogramming with Anti-miR223-Loaded Artificial Protocells Enhances In Vivo Cancer Therapeutic Potential.

Several immune cell-expressed miRNAs (miRs) are associated with altered prognostic outcome in cancer patients, suggesting that they may be potential targets for development of cancer therapies. Here, translucent zebrafish (Danio rerio) is utilized to demonstrate that genetic knockout or knockdown of one such miR, microRNA-223 (miR223), globally or specifically in leukocytes, does indeed lead to reduced cancer progression. As a first step toward potential translation to a clinical therapy, a novel strategy is described for reprogramming neutrophils and macrophages utilizing miniature artificial protocells (PCs) to deliver anti-miRs against the anti-inflammatory miR223. Using genetic and live imaging approaches, it is shown that phagocytic uptake of anti-miR223-loaded PCs by leukocytes in zebrafish (and by human macrophages in vitro) effectively prolongs their pro-inflammatory state by blocking the suppression of pro-inflammatory cytokines, which, in turn, drives altered immune cell-cancer cell interactions and ultimately leads to a reduced cancer burden by driving reduced proliferation and increased cell death of tumor cells. This PC cargo delivery strategy for reprogramming leukocytes toward beneficial phenotypes has implications also for treating other systemic or local immune-mediated pathologies.

Towards manufactured red blood cells for the treatment of inherited anemia.

Patients with inherited anemia and hemoglobinopathies (such as sickle cell disease and ß-thalassemia) are treated with red blood cell (RBC) transfusions to alleviate their symptoms. Some of these patients may have rare blood group types or go on to develop alloimmune reactions, which can make it difficult to source compatible blood in the donor population. Laboratory-grown RBC represent a particularly attractive alternative which could satisfy an unmet clinical need. The challenge, however, is to produce - from a limited number of stem cells - the 2x1012 RBC required for a standard adult therapeutic dose. Encouraging progress has been made in RBC production from adult stem cells under good manufacturing practice. In 2011, the Douay group conducted a successful proof-of-principle mini-transfusion of autologous manufactured RBC in a single volunteer. In the UK, a trial is planned to assess whether manufactured RBC are equivalent to RBC produced naturally in donors, by testing an allogeneic mini-dose of laboratory-grown manufactured RBC in multiple volunteers. This review discusses recent progress in the erythroid culture field as well as opportunities for further scaling up of manufactured RBC production for transfusion practice.

Ephrin/Eph receptor interaction facilitates macrophage recognition of differentiating human erythroblasts.

Erythropoiesis is one of the most efficient cellular processes in the human body producing approximately 2.5 million red blood cells every second. This process occurs in a bone marrow niche comprised of a central resident macrophage surrounded by differentiating erythroblasts, termed an erythroblastic island. It is not known what initially attracts the macrophage to erythroblasts to form these islands. The ephrin/Eph receptor family are known to regulate heterophilic cell-cell adhesion. We find that human VCAM1+ and VCAM1- bone marrow macrophages and in vitro cultured macrophages are ephrin-B2 positive, whereas differentiating human erythroblasts express EPHB4, EPHB6 and EPHA4. Furthermore, we detect a rise in integrin activation on erythroblasts at the stage at which the cells bind which is independent of EPH receptor presence. Using a live cell imaging assay, we show that specific inhibitory peptides or shRNA depletion of EPHB4 cause a significant reduction in the ability of macrophages to interact with erythroblasts but do not affect integrin activation. This study demonstrates for the first time that EPHB4 expression is required on erythroblasts to facilitate the initial recognition and subsequent interaction with macrophages, alongside the presence of active integrins.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.

Polyurethane scaffolds seeded with CD34(+) cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors.

We describe a 3D erythroid culture system that utilises a porous polyurethane (PU) scaffold to mimic the compartmentalisation found in the bone marrow. PU scaffolds seeded with peripheral blood CD34(+) cells exhibit a remarkable reproducibility of egress, with an increased output when directly compared to human bone scaffolds over 28 days. Immunofluorescence demonstrated the persistence of CD34(+) cells within the scaffolds for the entirety of the culture. To characterise scaffold outputs, we designed a flow cytometry panel that utilises surface marker expression observed in standard 2D erythroid and megakaryocyte cultures. This showed that the egress population is comprised of haematopoietic progenitor cells (CD36(+)GPA(-/low)). Control cultures conducted in parallel but in the absence of a scaffold were also generally maintained for the longevity of the culture albeit with a higher level of cell death. The harvested scaffold egress can also be expanded and differentiated to the reticulocyte stage. In summary, PU scaffolds can behave as a subtractive compartmentalised culture system retaining and allowing maintenance of the seeded "CD34(+) cell" population despite this population decreasing in amount as the culture progresses, whilst also facilitating egress of increasingly differentiated cells.