Use Of Macrophages Research Articles

Roles of Macrophages and Their Interactions with Schwann Cells After Peripheral Nerve Injury.

The adult peripheral nervous system has a significant ability for regeneration compared to the central nervous system. This is related to the unique neuroimmunomodulation after peripheral nerve injury (PNI). Unlike the repair of other tissues after injury, Schwann cells (SCs) respond immediately to the trauma and send out signals to precisely recruit macrophages to the injured site. Then, macrophages promote the degradation of the damaged myelin sheath by phagocytosis of local debris. At the same time, macrophages and SCs jointly secrete various cytokines to reconstruct a microenvironment suitable for nerve regeneration. This unique pathophysiological process associated with macrophages provides important targets for the repair and treatment of PNI, as well as an important reference for guiding the repair of other nerve injuries. To understand these processes more systematically, this paper describes the characteristics of macrophage activation and metabolism in PNI, discusses the underlying molecular mechanism of interaction between macrophages and SCs, and reviews the latest research progress of crosstalk regulation between macrophages and SCs. These concepts and therapeutic strategies are summarized to provide a reference for the more effective use of macrophages in the repair of PNI.

Biomimetic Systems Involving Macrophages and Their Potential for Targeted Drug Delivery.

The concept of targeted drug delivery can be described in terms of the drug systems' ability to mimic the biological objects' property to localize to target cells or tissues. For example, drug delivery systems based on red blood cells or mimicking some of their useful features, such as long circulation in stealth mode, have been known for decades. On the contrary, therapeutic strategies based on macrophages have gained very limited attention until recently. Here, we review two biomimetic strategies associated with macrophages that can be used to develop new therapeutic modalities: first, the mimicry of certain types of macrophages (i.e., the use of macrophages, including tumor-associated or macrophage-derived particles as a carrier for the targeted delivery of therapeutic agents); second, the mimicry of ligands, naturally absorbed by macrophages (i.e., the use of therapeutic agents specifically targeted at macrophages). We discuss the potential applications of biomimetic systems involving macrophages for new advancements in the treatment of infections, inflammatory diseases, and cancer.

A suspension of inactivated bacteria used for vaccination against recurrent urinary tract infections increases the phagocytic activity of murine macrophages.

Urinary tract infections are a major cause of the consumption of antibiotics in humans. We studied the effect of a vaccine (StroVac®, containing inactivated bacteria and used to prevent recurrent urinary tract infections) licensed in Germany on the release of pro-inflammatory cytokines and the phagocytosis of Escherichia (E.) coli in primary murine macrophages and the macrophage cell line J774A.1. StroVac® increased the release of the cytokines TNF-α, IL-6, IL-12/23 p40, and IL-1β and stimulated the phagocytosis of E. coli in a dose-dependent manner. This effect was independent of LPS as shown by the use of macrophages isolated from LPS-resistant C3H/HeJ mice. At concentrations up to 30 mg/l it was not toxic to bacteria or eukaryotic cells. StroVac® does not only act via the adaptive but also by stimulating the innate immune system. This stimulation may help to build trained innate immunity against bacterial pathogens involved in recurrent urinary tract infections.

Autophagic reprogramming of bone marrow–derived macrophages

Macro-autophagy is a highly conserved catabolic process among eukaryotes affecting macrophages. This work studies the genetic regulatory network involving the interplay between autophagy and macrophage polarization (activation). Autophagy-related genes (Atgs) and differentially expressed genes (DEGs) of macrophage polarization (M1–M2) were predicted, and their regulatory networks constructed. Naïve (M0) mouse bone marrow–derived monocytes were differentiated into M1 and M2a. Validation of the targets of Smad1, LC3A and LC3B, Atg16L1, Atg7, IL-6, CD68, Arg-1, and Vamp7 was performed in vitro. Immunophenotyping by flow cytometry revealed three macrophage phenotypes: M0 (IL-6 + /CD68 +), M1 (IL-6 + /CD68 + /Arg-1 +), and M2a (CD68 + /Arg-1). Confocal microscopy revealed increased autophagy in both M1 and M2a and a significant increase in the pre-autophagosomes size and number. Bafilomycin A increased the expression of CD68 and Arg-1 in all cell lineages. In conclusion, our approach predicted the protein targets mediating the interplay between autophagy and macrophage polarization. We suggest that autophagy reprograms macrophage polarization via CD68, arginase 1, Atg16L1-1, and Atg16L1-3. The current findings provide a foundation for the future use of macrophages in immunotherapy of different autoimmune disorders.

Hox-driven conditional immortalization of myeloid and lymphoid progenitors: Uses, advantages, and future potential.

Those who study macrophage biology struggle with the decision whether to utilize primary macrophages derived directly from mice or opt for the convenience and genetic tractability of immortalized macrophage-like cell lines in in vitro studies. Particularly when it comes to studying phagocytosis and phagosomal maturation-a signature cellular process of the macrophage-many commonly used cell lines are not representative of what occurs in primary macrophages. A system developed by Mark Kamps' group, that utilizes conditionally constitutive activity of Hox transcription factors (Hoxb8 and Hoxa9) to immortalize differentiation-competent myeloid cell progenitors of mice, offers an alternative to the macrophage/macrophage-like dichotomy. In this resource, we will review the use of Hoxb8 and Hoxa9 as hematopoietic regulators to conditionally immortalize murine hematopoietic progenitor cells which retain their ability to differentiate into many functional immune cell types including macrophages, neutrophils, basophils, osteoclasts, eosinophils, dendritic cells, as well as limited potential for the generation of lymphocytes. We further demonstrate that the use of macrophages derived from Hoxb8/Hoxa9 immortalized progenitors and their similarities to bone marrow-derived macrophages. To supplement the existing data, mass spectrometry-based proteomics, flow cytometry, cytology, and in vitro phagosomal assays were conducted on macrophages derived from Hoxb8 immortalized progenitors and compared to bone marrow-derived macrophages and the macrophage-like cell line J774. We additionally propose the use of a standardized nomenclature to describe cells derived from the Hoxb8/Hoxa9 system in anticipation of their expanded use in the study of leukocyte cell biology.

Abstract 502: In vitro characterization of antibodies targeting the SIRPα-CD47 phagocytosis checkpoint using tumor-associated macrophages from HuGEMM™ mice

Abstract Introduction: The signal-regulatory protein SIRPα-CD47 pathway is a phagocytosis checkpoint for macrophages and other innate immune cells. Tumor cells ubiquitously express CD47, and blocking SIRPα-CD47 interactions has been shown to promote cancer cell elimination by macrophage phagocytosis. The impact of SIRPα-CD47 blockade may be further improved by targeting prophagocytic receptors (Fc receptors) to enhance phagocytosis for anti-CD47 antibodies. Tumor-associated macrophages (TAMs) are the most abundant innate immune cells in late-stage tumors. Currently, drug development targeting CD47-SIRPα is rapidly growing, requiring robust in vitro macrophage/tumor cell co-culture assays to evaluate new agents targeting this pathway. Here, we report on the use of macrophages from an engineered mouse model expressing human SIRPα, which may serve as a good tool to test drug candidates. Methods: We have developed and characterized the human CD47 and SIRPα double knock-in mouse model (CD47/SIRPα HuGEMM) via CRISPR/Cas9 targeting. To characterize the effects of anti-human CD47 antibodies on phagocytosis and tumor cell killing, TAMs were isolated from tumor-bearing HuGEMM mice for in vitro macrophage/tumor cell co-culture. Results: Human CD47 expressing tumor cells, such as murine MC38-hCD47 and human Jurkat cells, were labeled with CFSE and used as target cells for antibody-dependent cellular phagocytosis (ADCP) and tumor cell death. The percentage of CFSE-positive among macrophages, and the percentage of dead cells among CFSE-positive tumor cell death, were measured by flow cytometry. We observed significant ADCP effects by TAM and killing of tumor cells after in vitro anti-CD47 antibody treatment. The contribution to ADCP by SIRPα-CD47 blockade and/or Fc effector function of antibodies was further investigated. We also evaluated the effect of SIRPα-CD47 blockade on the modulation of TAM phenotypes. Conclusion: Our data show that the CD47/SIRPα HuGEMM system provides an excellent tool for in vitro functional assays for drug candidates targeting the SIRPα-CD47 pathway, to evaluate the effect on ADCP by macrophages, activation of macrophages, and overall tumor cell death. Citation Format: Xuefei Yan, Hongjuan Zhang, Yi Liu, Yahong Zhang, Lei Zheng, Mingfa Zang, Annie Xiaoyu An, kevin Qijin Xu, Davy Ouyang, Henry Li, Yujun Huang. In vitro characterization of antibodies targeting the SIRPα-CD47 phagocytosis checkpoint using tumor-associated macrophages from HuGEMM™ mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 502.

The Role of Macrophages in Oral Squamous Cell Carcinoma

Oral cancer is a common malignancy worldwide, with high disease-related death rates. Oral squamous cell carcinoma (OSCC) accounts for more than 90% of oral tumors, with surgical management remaining the treatment of choice. However, advanced and metastatic OSCC is still incurable. Thus, emphasis has been given lately in understanding the complex role of the oral tumor microenvironment (TME) in OSCC progression, in order to identify novel prognostic biomarkers and therapeutic targets. Tumor associated macrophages (TAMs) constitute a major population of the OSCC TME, with bipolar role in disease progression depending on their activation status (M1 vs. M2). Here, we provide an up to date review of the current literature on the role of macrophages during oral oncogenesis, as well as their prognostic significance in OSCC survival and response to standard treatment regimens. Finally, we discuss novel concepts regarding the potential use of macrophages as targets for OSCC immunotherapeutics and suggest future directions in the field.

Macrophages as a Double-Edged Weapon: The Use of Macrophages in Cancer Immunotherapy and Understanding the Cross-Talk Between Macrophages and Cancer

Macrophages (Mϕs) play an essential role in maintaining body homeostasis. They perform dual functions produced by different subtypes. Mϕs not only fight against pathogens and foreign bodies such as bacteria or cancer cells but also participate in healing and repairing damaged tissue since they maintain both proinflammatory and anti-inflammatory effects sequentially. Tumors possess the ability to polarize Mϕs from proinflammatory M1 subtype to anti-inflammatory M2-like Mϕs called tumor-associated macrophages, which, in turn, help the tumors to acquire cancer hallmarks. Consequently, this polarization allows tumors to grow and spread. In this light, Mϕs have been a subject of intense study, and researchers have developed protocols to derive different Mϕs subtypes either as a new state-of-the-art therapeutic approach or to understand the cross-talk between cancer and Mϕs. In this review, we present the use of primary Mϕs in adoptive immunotherapy for cancer, illustrate the reciprocating interplay between cancer and Mϕs, and the resulting structural and functional change on both cell types. Furthermore, we summarize the recent cutting-edge approaches of using Mϕs in cancer immunotherapy.

Selenium-dependent metabolic reprogramming during inflammation and resolution

Trace element selenium (Se) is incorporated as the 21st amino acid, selenocysteine, into selenoproteins through tRNA[Ser]Sec. Selenoproteins act as gatekeepers of redox homeostasis and modulate immune function to effect anti-inflammation and resolution. However, mechanistic underpinnings involving metabolic reprogramming during inflammation and resolution remain poorly understood. Bacterial endotoxin lipopolysaccharide (LPS) activation of murine bone marrow–derived macrophages cultured in the presence or absence of Se (as selenite) was used to examine temporal changes in the proteome and metabolome by multiplexed tandem mass tag–quantitative proteomics, metabolomics, and machine-learning approaches. Kinetic deltagram and clustering analysis indicated that addition of Se led to extensive reprogramming of cellular metabolism upon stimulation with LPS enhancing the pentose phosphate pathway, tricarboxylic acid cycle, and oxidative phosphorylation, to aid in the phenotypic transition toward alternatively activated macrophages, synonymous with resolution of inflammation. Remodeling of metabolic pathways and consequent metabolic adaptation toward proresolving phenotypes began with Se treatment at 0 h and became most prominent around 8 h after LPS stimulation that included succinate dehydrogenase complex, pyruvate kinase, and sedoheptulokinase. Se-dependent modulation of these pathways predisposed bone marrow–derived macrophages to preferentially increase oxidative phosphorylation to efficiently regulate inflammation and its timely resolution. The use of macrophages lacking selenoproteins indicated that all three metabolic nodes were sensitive to selenoproteome expression. Furthermore, inhibition of succinate dehydrogenase complex with dimethylmalonate affected the proresolving effects of Se by increasing the resolution interval in a murine peritonitis model. In summary, our studies provide novel insights into the role of cellular Se via metabolic reprograming to facilitate anti-inflammation and proresolution.

Use of macrophage as a Trojan horse for cancer nanotheranostics

Nano-based cancer theranostics are often not as effective as expected due to the low accumulation of nanoparticles in tumors, and cell-aided strategies may represent a rescue to improve the efficacy of nanomedicines. Among the strategies, macrophages appear to be an outstanding candidate due to their wide-spread tissue presence and natural ability to take up nanoparticles carrying cargoes, and also the remarkable capability to modulate tumor microenvironment for enhanced therapeutic efficacy and anti-tumor immunotherapy. However, there are limitations, inconsistencies, and knowledge gaps in the current stage in developing macrophage-mediated nanotherapeutics to realize its fullest potential. Thus, it demands more efforts on understanding the interplays between macrophage and nanomedicine as well as nanomedicines and tumor cells in modulating anti-cancer immunity in the tumor microenvironment. Here, we recapitulate the current progress and knowledge gaps in the macrophage-mediated nano-drug delivery system, and we also shed light on future research directions towards clinical translation.

Host-Microbial Gut Interactions and Mushroom Nutrition

There is a tremendous complexity of the human gut microbiota in both health and disease states and a healthy microbiota consists of an inter-dependent network of microbes rather than a particular bacterial genera. The microbiota of the gastrointestinal tract is a symbiotic partner of the host as it is crucial for maintaining homeostasis and multiple components of the host immune system. Numerous host factors influence the composition of the microbiota early in life including diet, hygiene, environmental contacts, antibiotic use, and breastfeeding. Although the content of any diet can effect bacterial composition, it cannot be suggested that diet alone is responsible for the diversity of the microbiota or its variation among individuals. The intestinal physical-chemical barrier forms part of the intestinal immune system and plays a critical role in determining the composition of the microbiota. There are multiple recognised clinical uses of mushrooms due to their content in β-glucans, important antioxidant and cytoprotective enzymes, secondary metabolites and still other unknown factors. Mushroom β-glucans have been proposed to act as "biological response modifiers" based on their effects on the immune system, enhancing the body's own use of macrophages and T-lymphocytes, rather than directly attacking any tumours, controlling oxidative stress and inflammation.

Delivery of Pancreatic Islets and Single Dose Local Immune Suppression Into Subcutaneous Space Using Injectable Hydrogel Provides Indefinite Survival of the Graft in Mouse Model of Diabetes

Introduction Pancreatic islet transplantation is a promising technique to treat type 1 diabetes. Long-term survival of the graft is required for a successful islet transplantation. Repeated use of immunosuppressive drugs after organ/cell transplantation often leads to severe adverse effects including nephrotoxicity, hepatotoxicity, and opportunistic infections. Thus, development of a local immunosuppression protocol is necessary to improve the islet graft survival in clinics. Materials and Methods Pancreatic islets from Sprague-Dawley rats were transplanted into the subcutaneous space of B6 mice using injectable hydrogel. Three major groups were prepared for transplantation. (1) Islets transplanted FK506-loaded poly(lactic-co-glycolic acid) microspheres (10 mg/kg), (2) Islets transplanted with clodronate liposomes (6.25 mg/kg), and (3) Islet transplanted with the combination of both immune suppressants. The suspension of islets and immune suppressants in Matrigel was then injected into the subcutaneous space over the flanks of streptozocin-induced diabetic mice. Results Islets transplanted without any immunosuppression were rejected within two weeks. In contrast, the islets transplanted with the single immunosuppressive regimen of FK506 or clodronate improved survival rate compared with that of the control mice. More interestingly, the graft transplanted using the combination of both immune suppressants survived indefinitely. Immunological studies revealed that the immunosuppressive cocktail inhibited the proliferation of immune cells residing at the peripheral lymph nodes. Interestingly, the systemic immune system of the transplanted mice remained unaffected. Furthermore, histochemical analysis revealed the intact morphology of the islets at the transplanted site when codelivered with the immunosuppressant. Discussion Antigen presenting cells and T-cells orchestrate the immune rejection cascade. Macrophage depletion by the liposomal clodronate and the inhibition of T-cell activation by FK506 completely blocked the immune rejection cascade in the immune competent mice. The inhibition of immune stimulation in the peripheral lymph nodes improved the islet grafted into the subcutaneous space. Thus, the use of local immune suppression is an effective approach to enhance the survival of the transplanted islets. Conclusion We developed a protocol for the local codelivery of pancreatic islets and immune suppressive agents. Indefinite graft survival was obtained with the use of macrophage depleting agent and T-cell inhibitor. The single dose of local immune suppression during transplantation may avoid toxic effects associated with a long-term use of immune suppressive agents in clinics. National Research Foundation of Korea (NRF) Grant nos: 2015R1A5A2009124 and 2017R1D1A1B03027831. Korea Health Industry Development Institute (KHIDI) Grant no: HI16C1767.

Development of a Highly Effective Protocol using Local Immune Suppression Strategy in Pancreatic Islet Transplantation

Introduction Pancreatic islet transplantation is a promising technique to treat type 1 diabetes. Long-term survival of the graft is required for a successful islet transplantation. Repeated use of immunosuppressive drugs after organ/cell transplantation often leads to severe adverse effects including nephrotoxicity, hepatotoxicity, and opportunistic infections. Thus, development of a local immunosuppression protocol is necessary to improve the islet graft survival in clinics. Materials and Methods Pancreatic islets from Sprague-Dawley rats were transplanted into the subcutaneous space of B6 mice using injectable hydrogel. Briefly, 1000 islet equivalents were suspended in Matrigel premixed with FK506-loaded poly(lactic-co-glycolic acid) microspheres (10 mg/kg) and clodronate liposomes (6.25 mg/kg) and injected into the subcutaneous space over the flanks of streptozocin-induced diabetic mice. Blood glucose level was monitored using a potable glucometer. Results Islets transplanted without any immunosuppression were rejected within two weeks. In contrast, the islets transplanted with the immunosuppressive regimen of FK506 and clodronate survived indefinitely. Immunological studies revealed that the immunosuppressive cocktail inhibited the proliferation of immune cells residing at the peripheral lymph nodes. Interestingly, the systemic immune system of the transplanted mice remained unaffected. Furthermore, histochemical analysis revealed the intact morphology of the islets at the transplanted site when codelivered with the immunosuppressant. Discussion Antigen presenting cells and T-cells orchestrate the immune rejection cascade. Macrophage depletion by the liposomal clodronate and the inhibition of T-cell activation by FK506 completely blocked the immune rejection cascade in the immune competent mice. The inhibition of immune stimulation in the peripheral lymph nodes improved the islet grafted into the subcutaneous space. Thus, the use of local immune suppression is an effective approach to enhance the survival of the transplanted islets. Conclusion We developed a protocol for the local codelivery of pancreatic islets and immune suppressive agents. Indefinite graft survival was obtained with the use of macrophage depleting agent and T-cell inhibitor. The single dose of local immune suppression during transplantation may avoid toxic effects associated with a long-term use of immune suppressive agents in clinics. National Research Foundation of Korea (NRF) Grant nos: 2015R1A5A2009124 and 2017R1D1A1B03027831. Korea Health Industry Development Institute (KHIDI) Grant no: HI16C1767.

Macrophages as Drug Delivery Carriers for Acoustic Phase-Change Droplets

The major challenges in treating malignant tumors are transport of therapeutic agents to hypoxic regions and real-time assessment of successful drug release via medical imaging modalities. In this study, we propose the use of macrophages (RAW 264.7 cells) as carriers of drug-loaded phase-change droplets to penetrate ischemic or hypoxic regions within tumors. The droplets consist of perfluoropentane, lipid and the chemotherapeutic drug doxorubicin (DOX, DOX-droplets). The efficiency of DOX-droplet uptake, migration mobility and viability of DOX-droplet-loaded macrophages (DLMs) were measured using a transmembrane cell migration assay, the alamarBlue assay and flow cytometric analysis, respectively. Our results indicate the feasibility of utilizing macrophages as DOX-droplet carriers (DOX payload of DOX-droplets: 459.3 ± 35.8 µg/mL, efficiency of cell uptake DOX-droplets: 88.8 ± 3.5%). The migration mobility (total number of migrated microphages) of DLMs decreased to 32.3% compared with that of healthy macrophages, but the DLMs provided contrast-enhanced ultrasound imaging (1.7-fold enhancement) and anti-tumor effect (70.9% cell viability) after acoustic droplet vaporization, suggesting the potential theranostic applications of DLMs. Future work will assess the tumor penetration ability of DLMs, mechanical effect of droplet vaporization on in vivo anti-tumor therapy and the release of the carried drug by ultrasound-triggered vaporization.

Lipidomics reveals physiological isotope effects during the enzymatic oxygenation of polyunsaturated fatty acids ex vivo.

Arachidonic acid (AA, 20:4) is an omega‐6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis‐allylic carbons within 1,4‐cis, cis‐diene units. Replacing bis‐allylic hydrogen atoms with deuterium has been shown to strongly slow the rate of enzymatic oxygenation of linoleic acid by soybean lipoxygenase, resulting in a kinetic isotope effect (KIE). Yet, experimental determination of the KIE during oxygenation of AA by mammalian cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on in vitro systems using purified enzymes and were limited by availability of deuterated PUFAs. Here we demonstrate the use of macrophages as an ex vivo model system to study the physiological kinetic isotope effect (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AA isotopologues. By extending sensitive lipidomic UPLC‐MS/MS approaches to simultaneously quantify native and deuterated lipid products, we were able to measure the magnitude of the PKIE for COX and LOX oxygenation of AA by macrophages. This work provides the first demonstration that hydrogen(s) present at carbon 10 (C10) of AA play an important role in the catalysis of prostaglandin and thromboxane synthesis. Furthermore, we discovered that deuteration of C10 also promotes the formation of the resolving lipid mediator lipoxin B4 by activated macrophages, possibly by interfering with AA cyclization and shunting AA toward LOX metabolism under physiological conditions.Support or Funding InformationNIH RO1 GM20501‐41This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

M0 and M2 Macrophages Enhance Vascularization of Tissue Engineering Scaffolds

In order to address limitations in the field of tissue engineering, vascularization of scaffolds has been considered. This work focuses on the use of macrophages as a novel cell source to enhance vessel formation within a 3D hydrogel scaffold. The influence macrophages can have on vessel development is generally believed to be dependent on macrophage phenotype. To study the influence of macrophage phenotypes in vessel development, we encapsulated endothelial cells and macrophages into a bioactive poly(ethylene glycol) (PEG)-based hydrogel. Specifically, the hydrogel was modified to be cell-adhesive and sensitive to proteolytic degradation. This hydrogel served as a controlled 3D matrix that can support the spontaneous formation of vascular tubule structures. When co-encapsulated with primary aortic endothelial cells (AECs) in the 3D hydrogel matrix, M2 macrophages enhanced the volume of tubules formed from AECs by nearly threefold when compared to AECs alone. M0 macrophages encapsulated with AECs also enhanced tubule volume by twofold when compared to AECs alone. Conversely, M1 macrophages encapsulated with AECs reduced tubule volume by nearly 90% of the tubule volume observed for AECs alone. We found that M2 and M0 macrophages were more likely to directly interact with endothelial cells within the PEG-based hydrogels than M1 macrophages. This data indicated that M2 macrophages can play a role in enhancing vessel formation. The field of tissue engineering endeavors to create tissues by seeding cells within 3D biodegradable scaffolds. The size of engineered tissue scaffolds is limited due to oxygen and nutrient demands. In order to overcome size limits, blood vessels can be grown in engineered tissues. This work focuses on using macrophages as a cell source to help vessel formation in a scaffold material. Macrophages are present at sites of implantations and are known to influence vessel formation. Macrophages can adopt different characteristics based on environmental cues. We investigate which macrophage characteristics allow macrophages to help vessel formation in engineered tissues.

Lipidomics Reveals Dramatic Physiological Kinetic Isotope Effects during the Enzymatic Oxygenation of Polyunsaturated Fatty Acids Ex Vivo.

Arachidonic acid (AA, 20:4) is an omega-6 polyunsaturated fatty acid (PUFA) and the main precursor to the class of lipid mediators known as eicosanoids. The enzymes that catalyze the oxygenation of AA begin by abstracting hydrogen from one of three bis-allylic carbons within 1,4-cis,cis-diene units. Substitution of deuterium for hydrogen has been shown to lead to massive kinetic isotope effects (KIE) for soybean lipoxygenase (sLOX) oxygenation of linoleic acid (LA, 18:2). Yet, experimental determination of the KIE during oxygenation of AA and LA by mammalian enzymes including cyclooxygenase (COX) and lipoxygenase (LOX) has revealed far lower values. All prior studies investigating the KIE of PUFA oxygenation have relied on in vitro systems using purified enzymes and were limited by availability of deuterated substrates. Here we demonstrate the use of macrophages as an ex vivo model system to study the physiological KIE (PKIE) during enzymatic AA oxygenation by living cells using a newly synthesized library of deuterated AA isotopologues. By extending lipidomic UPLC-MS/MS approaches to simultaneously quantify native and deuterated lipid products, we were able to demonstrate that the magnitude of the PKIE measured in macrophages for COX and LOX oxygenation of AA is similar to KIEs determined in previous reports using the AA isotopologue deuterated at carbon 13 (C13). However, for the first time we show that increasing the number of deuterated bis-allylic carbons to include both C10 and C13 leads to a massive increase in the PKIE for COX oxygenation of AA. We provide evidence that hydrogen(s) present at C10 of AA play a critical role in the catalysis of prostaglandin and thromboxane synthesis. Furthermore, we discovered that deuteration of C10 promotes the formation of the resolving lipid mediator lipoxin B4, likely by interfering with AA cyclization and shunting AA to the LOX pathway under physiological conditions.

Identifying exposition to low oxygen environment in human macrophages using secondary ion mass spectrometry and multivariate analysis.

Macrophages are innate immune cells presenting a strong phenotypic plasticity and deeply involved in tissue homeostasis. Oxygen environmental tension is a physical parameter that could influence their polarizations. In this study we use time-of-flight secondary ion mass spectrometry (TOF-SIMS) to describe how various polarizations are modified by a low oxygen exposure. TOF-SIMS experiments were performed using an IONTOF ToF-SIMS 5-100 (ION-TOF GmbH, Munster, Germany). Analysis was performed using a pulsed 25keV Bi3+ beam, sputtering was performed using a 250eV Cs beam. Cells were fixed by paraformaldehyde before TOF-SIMS analysis. Multivariate analysis of the TOF-SIMS spectra provided ion species associated with the exposure of macrophages to low oxygen concentration. We were able to obtain some species, specific of a particular polarization, advocating for the use of macrophages as reporter cells of oxygen tension in tissues. Our study demonstrates that macrophage molecular signature to low oxygen environment is dependent on their polarization. TOF-SIMS shows the clear capability to produce species revealing this exposition. This result opens the way to the use of TOF-SIMS as a tool to explore hypoxia in human tissues.

Nanomedicines for the Treatment of CNS Diseases.

Targeting and delivering macromolecular therapeutics to the central nervous system (CNS) has been a major challenge. The blood-brain barrier (BBB) is the main obstacle that must be overcome to allow compounds to reach their targets in the brain. Therefore, much effort has been channelled into improving transport of therapeutics across the BBB and into the CNS including the use of nanoparticles. In this thematic issue, several reviews and original research are presented that address "Nanomedicines for CNS Diseases." The articles in this issue are concentrated on either CNS-HIV disease or CNS tumors. In regards to CNS-HIV disease, there are two reviews that discuss the role of nanoparticles for improving the delivery of HIV therapeutics to the CNS. In addition, there are two original articles focusing on therapies for CNS-HIV, one of them uses nanoparticles for delivery of siRNA specific to a key protein in autophagy to microglia, and another discusses nanoparticle delivery of a soluble mediator to suppress neuroinflammation. Furthermore, a comprehensive review about gene therapy for CNS neurological diseases is also included. Finally, this issue also includes review articles on enhanced drug targeting to CNS tumors. These articles include a review on the use of nanoparticles for CNS tumors, a review on functionalization (ligands) of nanoparticles for drug targeting to the brain tumor by overcoming BBB, and the final review discusses the use of macrophages as a delivery vehicle to CNS tumors. This thematic issue provides a wealth of knowledge on using nanomedicines for CNS diseases.

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry.

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal disease. Macrophages are heterogeneous cells that have been implicated in kidney injury. Macrophages may be classified into two different phenotypes: classically activated macrophages (M1 macrophages), that release pro-inflammatory cytokines and promote fibrosis; and alternatively activated macrophages (M2 macrophages) that are associated with immunoregulatory and tissue-remodeling functions. These macrophage phenotypes need to be discriminated and analyzed to determine their contribution to renal injury. However, there are scarce studies reporting consistent phenotypic and functional information about macrophage subtypes in inflammatory renal disease models, especially in rats. This fact may be related to the limited macrophage markers used in rats, contrary to mice. Therefore, novel strategies are necessary to quantify and characterize the renal content of these infiltrating cells in a reliable way. This manuscript details a protocol for kidney digestion and further phenotypic and quantitative analysis of macrophages from rat kidneys by flow cytometry. Briefly, kidneys were incubated with collagenase and total macrophages were identified according to the dual presence of CD45 (leukocytes common antigen) and CD68 (PAN macrophage marker) in live cells.This was followed by surface staining of CD86 (M1 marker) and CD163 (M2 marker). Rat peritoneal macrophages were used as positive control for macrophage marker detection by flow cytometry. Our protocol resulted in low cellular mortality and allowed characterization of different intracellular and surface protein markers, thus limiting the loss of cellular integrity observed in other protocols. Moreover, this procedure allows the use of macrophages for further techniques, including cell sorting and mRNA or protein expression studies, among others.