Regenerative Macrophages Research Articles

Magnetic soft microrobots for erectile dysfunction therapy

Erectile dysfunction (ED) is a major threat to male fertility and quality of life, and mesenchymal stromal cells (MSCs) are a promising therapeutic option. However, therapeutic outcomes are compromised by low MSC retention and survival rates in corpus cavernosum tissue. Here, we developed an innovative magnetic soft microrobot comprising an ultrasoft hydrogel microsphere embedded with a magnetic nanoparticle chain for MSC delivery. This design also features phenylboronic acid groups for scavenging reactive oxygen species (ROS). With a Young's modulus of less than 1 kPa, the ultrasoft microrobot adapts its shape within narrow blood vessels, ensuring a uniform distribution of MSCs within the corpus cavernosum. Our findings showed that compared with traditional MSC injections, the MSC delivery microrobot (MSC-Rob) significantly enhanced MSC retention and survival. In both rat and beagle ED models, MSC-Rob treatment accelerated the repair of corpus cavernosum tissue and restored erectile function. Single-cell RNA sequencing (scRNA-seq) revealed that MSC-Rob treatment facilitates nerve and blood vessel regeneration in the corpus cavernosum by increasing the presence of regenerative macrophages. Overall, our MSC-Rob not only advances the clinical application of MSCs for ED therapy but also broadens the scope of microrobots for other cell therapies.

Delayed administration of interleukin-4 coacervate alleviates the neurotoxic phenotype of astrocytes and promotes functional recovery after a contusion spinal cord injury

Objective. Macrophages and astrocytes play a crucial role in the aftermath of a traumatic spinal cord injury (SCI). Infiltrating macrophages adopt a pro-inflammatory phenotype while resident astrocytes adopt a neurotoxic phenotype at the injury site, both of which contribute to neuronal death and inhibit axonal regeneration. The cytokine interleukin-4 (IL-4) has shown significant promise in preclinical models of SCI by alleviating the macrophage-mediated inflammation and promoting functional recovery. However, its effect on neurotoxic reactive astrocytes remains to be elucidated, which we explored in this study. We also studied the beneficial effects of a sustained release of IL-4 from an injectable biomaterial compared to bolus administration of IL-4.Approach. We fabricated a heparin-based coacervate capable of anchoring and releasing bioactive IL-4 and tested its efficacyin vitroandin vivo. Main results. We show that IL-4 coacervate is biocompatible and drives a robust anti-inflammatory macrophage phenotype in culture. We also show that IL-4 and IL-4 coacervate can alleviate the reactive neurotoxic phenotype of astrocytes in culture. Finally, using a murine model of contusion SCI, we show that IL-4 and IL-4 coacervate, injected intraspinally 2 d post-injury, can reduce macrophage-mediated inflammation, and alleviate neurotoxic astrocyte phenotype, acutely and chronically, while also promoting neuroprotection with significant improvements in hindlimb locomotor recovery. We observed that IL-4 coacervate can promote a more robust regenerative macrophage phenotypein vitro, as well as match its efficacyin vivo,compared to bolus IL-4.Significance. Our work shows the promise of coacervate as a great choice for local and prolonged delivery of cytokines like IL-4. We support this by showing that the coacervate can release bioactive IL-4, which acts on macrophages and astrocytes to promote a pro-regenerative environment following a SCI leading to robust neuroprotective and functional outcomes.

Role of muscle regenerative macrophages in adjuvant-mediated immune response skewing to seasonal influenza vaccine

Abstract MF59 (AddaVax-like) is a clinically approved nanoemulsion adjuvant with an unclear mechanism of action. Here we introduce a novel approach to investigating the role of muscle regeneration in adjuvanted vaccine response. We found that AddaVax adjuvanted quadrivalent inactivated flu vaccine (QIV) induced a Th2 immune response with IL-4, -5, and -13 expression in the lungs 5 days after H1N1 lethal challenge. In contrast, IMDQ-PC (TLR7/8 agonist) induced a Th1 lung response and higher IgG2a titers. In BMDM cultures, we found AddaVax unlike IMDQ-PC to require IFNγ co-incubation for the induction of iNOS+ proinflammatory M1 phenotype. Since IFNγ is crucial for muscle regeneration, we hypothesized that AddaVax triggers macrophage-dependent muscle regeneration which leads to an enhanced Th2 vaccine response. We found that AddaVax adjuvanted QIV led to an increase of Arg-I+ M2 macrophages in the muscle 4 days post-injection, similar to late-stage Th2 response in regeneration. Using IgG titers, Our hypothesis was further supported by finding AddaVax response to be diminished in aged mice where regeneration is impaired, but rescuable by depletion of circulating monocytes and reconstitution with young BMDMs. Using in-depth characterization and selective depletion, we will investigate the crosstalk between muscle regeneration and AddaVax response allowing a better understanding of the potential muscle role in inhibiting the Th1 response necessary in viral infections, and cancer vaccines.

Efficacy of HDAC Inhibitors in Driving Peroxisomal β-Oxidation and Immune Responses in Human Macrophages: Implications for Neuroinflammatory Disorders.

Elevated levels of saturated very long-chain fatty acids (VLCFAs) in cell membranes and secreted lipoparticles have been associated with neurotoxicity and, therefore, require tight regulation. Excessive VLCFAs are imported into peroxisomes for degradation by β-oxidation. Impaired VLCFA catabolism due to primary or secondary peroxisomal alterations is featured in neurodegenerative and neuroinflammatory disorders such as X-linked adrenoleukodystrophy and multiple sclerosis (MS). Here, we identified that healthy human macrophages upregulate the peroxisomal genes involved in β-oxidation during myelin phagocytosis and pro-inflammatory activation, and that this response is impaired in peripheral macrophages and phagocytes in brain white matter lesions in MS patients. The pharmacological targeting of VLCFA metabolism and peroxisomes in innate immune cells could be favorable in the context of neuroinflammation and neurodegeneration. We previously identified the epigenetic histone deacetylase (HDAC) inhibitors entinostat and vorinostat to enhance VLCFA degradation and pro-regenerative macrophage polarization. However, adverse side effects currently limit their use in chronic neuroinflammation. Here, we focused on tefinostat, a monocyte/macrophage-selective HDAC inhibitor that has shown reduced toxicity in clinical trials. By using a gene expression analysis, peroxisomal β-oxidation assay, and live imaging of primary human macrophages, we assessed the efficacy of tefinostat in modulating VLCFA metabolism, phagocytosis, chemotaxis, and immune function. Our results revealed the significant stimulation of VLCFA degradation with the upregulation of genes involved in peroxisomal β-oxidation and interference with immune cell recruitment; however, tefinostat was less potent than the class I HDAC-selective inhibitor entinostat in promoting a regenerative macrophage phenotype. Further research is needed to fully explore the potential of class I HDAC inhibition and downstream targets in the context of neuroinflammation.

Comparative single-cell profiling reveals distinct cardiac resident macrophages essential for zebrafish heart regeneration.

Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (-1d_CL, macrophage-delayed model) impairs neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first week post injury (Lai et al., 2017). It is thus intriguing to learn the regenerative macrophage property by comparing these late macrophages vs. control macrophages during cardiac repair. Here, we further investigate the mechanistic insights of heart regeneration by comparing the non-regenerative macrophage-delayed model with regenerative controls. Temporal RNAseq analyses revealed that -1d_CL treatment led to disrupted inflammatory resolution, reactive oxygen species homeostasis, and energy metabolism during cardiac repair. Comparative single-cell RNAseq profiling of inflammatory cells from regenerative vs. non-regenerative hearts further identified heterogeneous macrophages and neutrophils, showing alternative activation and cellular crosstalk leading to neutrophil retention and chronic inflammation. Among macrophages, two residential subpopulations (hbaa+ Mac and timp4.3+ Mac 3) were enriched only in regenerative hearts and barely recovered after +1d_CL treatment. To deplete the resident macrophage without delaying the circulating macrophage recruitment, we established the resident macrophage-deficient model by administrating CL earlier at 8 d (-8d_CL) before cryoinjury. Strikingly, resident macrophage-deficient zebrafish still exhibited defects in revascularization, cardiomyocyte survival, debris clearance, and extracellular matrix remodeling/scar resolution without functional compensation from the circulating/monocyte-derived macrophages. Our results characterized the diverse function and interaction between inflammatory cells and identified unique resident macrophages prerequisite for zebrafish heart regeneration.

Mesenchymal “stem” cells, or facilitators for the development of regenerative macrophages? Pericytes at the interface of wound healing

Cultured mesenchymal stromal cells are among the most used cells in clinical trials. Currently, their potential benefits include provision of mature cell types through differentiation, and secretion of various types of paracrine signaling molecules. Even though research on these cells has spanned some decades now, surprisingly, their therapeutic potential has not been fully translated into clinical practice yet, which calls for further understanding of their intrinsic nature and modes of action. In this review, after discussing pieces of evidence that suggest that some perivascular cells may exhibit mesenchymal stem cell characteristics in vivo, we examine the possibility that subpopulations of perivascular and/or adventitial cells activated after tissue injury behave as MSCs and contribute to the resolution of tissue injury by providing cues for the development of regenerative macrophages at injured sites. Under this perspective, an important contribution of cultured MSCs (or their acellular products, such as extracellular vesicles) used in cell therapies would be to instigate the development of M2-like macrophages that support the tissue repair process.

Autologous Platelet-Rich Growth Factor Reduces M1 Macrophages and Modulates Inflammatory Microenvironments to Promote Sciatic Nerve Regeneration.

The failure of peripheral nerve regeneration is often associated with the inability to generate a permissive molecular and cellular microenvironment for nerve repair. Autologous therapies, such as platelet-rich plasma (PRP) or its derivative platelet-rich growth factors (PRGF), may improve peripheral nerve regeneration via unknown mechanistic roles and actions in macrophage polarization. In the current study, we hypothesize that excessive and prolonged inflammation might result in the failure of pro-inflammatory M1 macrophage transit to anti-inflammatory M2 macrophages in large nerve defects. PRGF was used in vitro at the time the unpolarized macrophages (M0) macrophages were induced to M1 macrophages to observe if PRGF altered the secretion of cytokines and resulted in a phenotypic change. PRGF was also employed in the nerve conduit of a rat sciatic nerve transection model to identify alterations in macrophages that might influence excessive inflammation and nerve regeneration. PRGF administration reduced the mRNA expression of tumor necrosis factor-α (TNFα), interleukin-1β (IL-1β), and IL-6 in M0 macrophages. Increased CD206 substantiated the shift of pro-inflammatory cytokines to the M2 regenerative macrophage. Administration of PRGF in the nerve conduit after rat sciatic nerve transection promoted nerve regeneration by improving nerve gross morphology and its targeted gastrocnemius muscle mass. The regenerative markers were increased for regrown axons (protein gene product, PGP9.5), Schwann cells (S100β), and myelin basic protein (MBP) after 6 weeks of injury. The decreased expression of TNFα, IL-1β, IL-6, and CD68+ M1 macrophages indicated that the inflammatory microenvironments were reduced in the PRGF-treated nerve tissue. The increase in RECA-positive cells suggested the PRGF also promoted angiogenesis during nerve regeneration. Taken together, these results indicate the potential role and clinical implication of autologous PRGF in regulating inflammatory microenvironments via macrophage polarization after nerve transection.

Single-Cell RNA-seq Analysis Reveals Cellular Functional Heterogeneity in Dermis Between Fibrotic and Regenerative Wound Healing Fates.

BackgroundFibrotic scars are common in both human and mouse skin wounds. However, wound-induced hair neogenesis in the murine wounding models often results in regenerative repair response. Herein, we aimed to uncover cellular functional heterogeneity in dermis between fibrotic and regenerative wound healing fates.MethodsThe expression matrix of single-cell RNA sequencing (scRNA-seq) data of fibrotic and regenerative wound dermal cells was filtered, normalized, and scaled; underwent principal components analysis; and further analyzed by Uniform Manifold Approximation and Projection (UMAP) for dimension reduction with the Seurat package. Cell types were annotated, and cell–cell communications were analyzed. The core cell population myofibroblast was identified and the biological functions of ligand and receptor genes between myofibroblast and macrophage were evaluated. Specific genes between fibrotic and regenerative myofibroblast and macrophage were identified. Temporal dynamics of myofibroblast and macrophage were reconstructed with the Monocle tool.ResultsAcross dermal cells, there were six cell types, namely, EN1-negative myofibroblasts, EN1-positive myofibroblasts, hematopoietic cells, macrophages, pericytes, and endothelial cells. Ligand and receptor genes between myofibroblasts and macrophages mainly modulated cell proliferation and migration, tube development, and the TGF-β pathway. Specific genes that were differentially expressed in fibrotic compared to regenerative myofibroblasts or macrophages were separately identified. Specific genes between fibrotic and regenerative myofibroblasts were involved in the mRNA metabolic process and organelle organization. Specific genes between fibrotic and regenerative macrophages participated in regulating immunity and phagocytosis. We then observed the underlying evolution of myofibroblasts or macrophages.ConclusionCollectively, our findings reveal that myofibroblasts and macrophages may alter the skin wound healing fate through modulating critical signaling pathways.

Abstract 267: Altered Macrophage Response Associated With Impaired Skeletal Muscle Regeneration In Murine Model Of Limb Ischemia

Introduction: Skeletal muscle regeneration in the ischemic limb is a complex complex process dependent on coordinated intercellular communication involving an array of cell types. These interactions are not well understood, and the development of novel regenerative therapies for peripheral arterial disease will require new insight into the cellular heterogeneity and intercellular signaling that occurs in the ischemic limb. Macrophages play a role in orchestrating critical events in muscle regeneration; hence we sought to characterize their transcriptional signature and identify candidate signaling pathways in the context of limb ischemia. Methods: We applied single-cell RNA sequencing (scRNA-seq) to skeletal muscle obtained from regenerative (C57BL/6) and non-regenerative (BALB/c) mouse limbs at multiple time points following ligation of the femoral artery. We used CellChat, a computation tool, to predict signaling pathway activity between macrophages and muscle satellite cells (MuSC) based on our single-cell data. Results: We identified 12 distinct macrophage populations in the regenerative and non-regenerative limb, including strain-specific macrophage clusters associated with the response to ischemia. Regenerative macrophages displayed a proliferative phenotype, while non-regenerative macrophages exhibited a persistent pro-inflammatory phenotype. Additionally, we identified several candidate signaling interactions between specific macrophage clusters and MuSCs. Notable potential interactions included enhanced FN1 to SDC4 and THBS1 to SDC4 signaling between regenerative macrophage and MuSCs, as well as OPN to CD44 signaling between non-regenerative macrophages and MuSCs. Conclusions: Our study maps the dynamic macrophage response in the ischemic limb at single-cell resolution. This provides a valuable resource to investigate macrophage-mediated mechanisms of skeletal muscle regeneration in response to limb ischemia.

Enrichment of Nanofiber Hydrogel Composite with Fractionated Fat Promotes Regenerative Macrophage Polarization and Vascularization for Soft-Tissue Engineering.

Fractionated fat has been shown to promote dermal regeneration; however, the use of fat grafting for reconstruction of soft-tissue defects is limited because of volume loss over time. The authors have developed a novel approach for engineering of vascularized soft tissue using an injectable nanofiber hydrogel composite enriched with fractionated fat. Fractionated fat was generated by emulsification of groin fat pads from rats and mixed in a 3:1 ratio with nanofiber hydrogel composite (nanofiber hydrogel composite with fractionated fat). Nanofiber hydrogel composite with fractionated fat or nanofiber hydrogel composite alone was placed into isolation chambers together with arteriovenous loops, which were subcutaneously implanted into the groin of rats (n = 8 per group). After 21 days, animals were euthanized and systemically perfused with ink, and tissue was explanted for histologic analysis. Immunofluorescent staining and confocal laser scanning microscopy were used to quantify CD34+ progenitor cell and macrophage subpopulations. Nanofiber hydrogel composite with fractionated fat tissue maintained its shape without shrinking and showed a significantly stronger functional vascularization compared to composite alone after 21 days of implantation (mean vessel count, 833.5 ± 206.1 versus 296.5 ± 114.1; p = 0.04). Tissue heterogeneity and cell count were greater in composite with fractionated fat (mean cell count, 49,707 ± 18,491 versus 9263 ± 3790; p = 0.005), with a significantly higher number of progenitor cells and regenerative CD163+ macrophages compared to composite alone. Fractionated fat-enriched nanofiber hydrogel composite transforms into highly vascularized soft tissue over 21 days without signs of shrinking and promotes macrophage polarization toward regenerative phenotypes. Enrichment of injectable nanofiber hydrogel composite with fractionated fat represents a promising approach for durable reconstruction of soft-tissue defects. The authors' approach for tissue engineering may ultimately lay the groundwork for clinically relevant applications with the goal of generating large volumes of vascularized soft tissue for defect reconstruction without donor site morbidity.

Obesity Prolongs the Inflammatory Response in Mice After Severe Trauma and Attenuates the Splenic Response to the Inflammatory Reflex.

Thoracic traumas with extra-thoracic injuries result in an immediate, complex host response. The immune response requires tight regulation and can be influenced by additional risk factors such as obesity, which is considered a state of chronic inflammation. Utilizing high-dimensional mass and regular flow cytometry, we define key signatures of obesity-related alterations of the immune system during the response to the trauma. In this context, we report a modification in important components of the splenic response to the inflammatory reflex in obese mice. Furthermore, during the response to trauma, obese mice exhibit a prolonged increase of neutrophils and an early accumulation of inflammation associated CCR2+CD62L+Ly6Chi monocytes in the blood, contributing to a persistent inflammatory phase. Moreover, these mice exhibit differences in migration patterns of monocytes to the traumatized lung, resulting in decreased numbers of regenerative macrophages and an impaired M1/M2 switch in traumatized lungs. The findings presented in this study reveal an attenuation of the inflammatory reflex in obese mice, as well as a disturbance of the monocytic compartment contributing to a prolonged inflammation phase resulting in fewer phenotypically regenerative macrophages in the lung of obese mice.

Break monopoly of polarization: CD301b+ macrophages play positive roles in osteoinduction of calcium phosphate ceramics

The pro-inflammatory M1 and anti-inflammatory M2 polarization macrophages were considered crucial during the immune regulation of bone regeneration. However, M2 polarization macrophages cannot strictly represent regenerative macrophage phenotypes. There is an urgent need to classify a reliable regenerative macrophage to guide the development of osteoimmune modulation and bone regeneration biomaterials. Herein, we report a new CD301b+ macrophage phenotype in the osteoinduction process of calcium phosphate ceramics. There were far more CD301b+ macrophages around biphasic calcium phosphate ceramics than those around β-tricalcium phosphate ceramics. Additionally, CD301b-deficient mice failed to form new bone around biphasic calcium phosphate ceramics. Moreover, macrophages stimulated by biphasic calcium phosphate ceramics were beneficial to osteogenic differentiation of mesenchymal stem cells. CD301b+ macrophages can activate the IGF1R/Akt/mTOR pathway of mesenchymal stem cells by IGF1 expression, thereby promoting osteogenic differentiation. Furthermore, transplanting the sorted CD301b+ macrophages could further facilitate the osteoinduction of biphasic calcium phosphate ceramics. These data highlighted the critical role of CD301b+ macrophages in the osteoinduction of calcium phosphate ceramics. It may provide a novel target for potential osteoimmune modulation in bone regeneration.

Mussel patterned with 4D biodegrading elastomer durably recruits regenerative macrophages to promote regeneration of craniofacial bone

Crosstalk between bone marrow mesenchymal stem cells (BMSCs) and macrophages plays vital role in bone healing. By investigating the mechanism of collagen membrane-guided bone regeneration, we found compact structure and rapid membrane degradation compromised the duration of M2 macrophages influx, which restricts the recruitment of BMSCs that is essential for bone healing. To tackle this issue, a biodegrading elastomeric compound consisting of poly(glycerol sebacate) (PGS) and polycaprolactone (PCL) was fabricated into hierarchically porous membrane. The rational design of 3D microstructure enabled sufficient polydopamine (PDA) coating. Without any addition of growth factors, the 3D-patterned PDA membrane enables early and durable influx of M2 macrophages, which in turn promotes BMSCs recruitment and osteogenic differentiation. Furthermore, 4D-morphing of the membrane fully regenerates the dome shaped calvarial bone as well as arc-shape bone in peri-implant alveolar defect without filling xenogenous substitute. This study revealed the superiority of 3D printed microstructures in immunomodulatory materials. The availability of 4D-morphing for PGS/PCL construct expanded their advantages in reconstructing craniofacial bone.

REGENERATIVE MACROPHAGES: A NEW HOPE FOR CARDIOMYOPATHY

Globally, cardiovascular diseases (CVD) are the number one cause of death. It is predicted that 23.6 million people will die from it by 2030, mainly caused by strokes and heart attacks. This study aimed to investigate the new treatment of cardiomyopathy, a cardiovascular disease that hinders blood supply from the heart to the body and can cause heart failure. While the traditional treatment focus on cardiac rehabilitation, including lifestyle counseling and prescription exercise, surgery on coronary bypass artery, and medication of increasing the flow of blood, the use of therapeutic cells is a new way to deal with cardiovascular diseases and heart infarction. It intends to effectively replace or regenerate dead or damaged heart cells, as the main advantage of this treatment. The use of alternative methods to treat cardiovascular diseases has high potential value, as found in the immunotherapy method through the use of "regenerative macrophages." This study is a qualitative case report research study which provides an exploration of phenomenon or opportunity of sharing efforts to intervene single patient’s health with unreported health features previously. The result of this study showed that a 71 years old man was hospitalized in Gatot Soebroto Central Army Hospital Jakarta, Indonesia, by the diagnosis of coronary artery disease. He presented for chronic cough and peripheral edema. He denied syncope, dyspnea, nausea, vomiting, and diaphoresis. The application of echocardiography regarding the previous treatment indicated diastolic dysfunction, left ventricle hypokinesis and left ventricular hypertrophy with 44% of low ejection on the fraction. After the vaccines of Regenerative Macrophages (ReM) therapy were given to the patient, significant improvements in the echocardiography parameters were observed, with normal kinetics, improved ejection fraction, and diastolic dysfunction increased to 56%. Significantly, regenerative macrophages could be an option of immunotherapy in patients with cardiomyopathies with low risk of effective treatment further.

Mechanisms of uptake of mesenchymal stromal cell membrane particles

Background & Aim Mesenchymal stromal cells (MSC) are a promising therapy for inflammatory diseases. Even so, culture-expanded MSC are large and get trapped in the pulmonary capillaries after intravenous infusion, where they have a short survival time. To steer cellular immunoregulatory therapy beyond the lungs, we generated nm-sized particles from MSC membranes (membrane particles, MP) which interact with immune cells and possess immune regulatory properties. However, the intracellular trafficking pathway of MP in recipient cells remains unclear. Methods, Results & Conclusion Here, we characterized MP uptake and internalization by macrophages and endothelial cells. For that, different endocytotic uptake mechanisms of MP, such as phagocytosis, macropinocytosis, dynamin-, clathrin- and caveolin-mediated endocytosis were investigated in human THP-1 macrophages and human umbilical vein endothelial cells (HUVEC) by flow cytometry and confocal microscopy. We demonstrated that MP uptake by macrophages and HUVEC is dose, time, size, and energy-dependent. Also, MP bind to cells through cell-surface receptors, such as heparan sulfate proteoglycans (HSPGs), and are subsequently be internalized. This recipient cell-MP interaction is partially inhibited in the presence of heparin, a soluble analogue of HSPGs. The polarization of M1 and M2 macrophages was achieved by stimulation with LPS and IFN-γ (proinflammatory condition) or IL-4 and IL-13 (anti-inflammatory condition), respectively. Thus, M2 cells exhibited greater endocytic capacity than M1 cells, suggesting that MP may act in particular on regulatory and regenerative macrophages. Under inflammatory stimuli, HUVEC displayed a similar capacity to MP internalization as under control conditions. Moreover, specific inhibitors for endocytotic pathways revealed that MP internalization by macrophages and HUVEC depends on dynamin- and clathrin-mediated endocytosis, but does not involve caveolae-mediated endocytosis. The actin cytoskeleton and phosphoinositide 3-kinase, which are implicated in macropinocytosis and phagocytosis, are also required in MP uptake by both macrophages and HUVEC. Taken together, these results suggest that MP enter macrophages and HUVEC via a variety of mechanisms, including dynamin- and clathrin-dependent endocytosis, caveolae-independent endocytosis, macropinocytosis, and phagocytosis. Identification of mechanisms involved in MP internalization under different conditions allows specific modulation of MP delivery to target cells.

Polycistronic Delivery of IL-10 and NT-3 Promotes Oligodendrocyte Myelination and Functional Recovery in a Mouse Spinal Cord Injury Model.

One million estimated cases of spinal cord injury (SCI) have been reported in the United States and repairing an injury has constituted a difficult clinical challenge. The complex, dynamic, inhibitory microenvironment postinjury, which is characterized by proinflammatory signaling from invading leukocytes and lack of sufficient factors that promote axonal survival and elongation, limits regeneration. Herein, we investigated the delivery of polycistronic vectors, which have the potential to coexpress factors that target distinct barriers to regeneration, from a multiple channel poly(lactide-co-glycolide) (PLG) bridge to enhance spinal cord regeneration. In this study, we investigated polycistronic delivery of IL-10 that targets proinflammatory signaling, and NT-3 that targets axonal survival and elongation. A significant increase was observed in the density of regenerative macrophages for IL-10+NT-3 condition relative to conditions without IL-10. Furthermore, combined delivery of IL-10+NT-3 produced a significant increase of axonal density and notably myelinated axons compared with all other conditions. A significant increase in functional recovery was observed for IL-10+NT-3 delivery at 12 weeks postinjury that was positively correlated to oligodendrocyte myelinated axon density, suggesting oligodendrocyte-mediated myelination as an important target to improve functional recovery. These results further support the use of multiple channel PLG bridges as a growth supportive substrate and platform to deliver bioactive agents to modulate the SCI microenvironment and promote regeneration and functional recovery. Impact statement Spinal cord injury (SCI) results in a complex microenvironment that contains multiple barriers to regeneration and functional recovery. Multiple factors are necessary to address these barriers to regeneration, and polycistronic lentiviral gene therapy represents a strategy to locally express multiple factors simultaneously. A bicistronic vector encoding IL-10 and NT-3 was delivered from a poly(lactide-co-glycolide) bridge, which provides structural support that guides regeneration, resulting in increased axonal growth, myelination, and subsequent functional recovery. These results demonstrate the opportunity of targeting multiple barriers to SCI regeneration for additive effects.

Microchannel network hydrogel induced ischemic blood perfusion connection

Angiogenesis induction into damaged sites has long been an unresolved issue. Local treatment with pro-angiogenic molecules has been the most common approach. However, this approach has critical side effects including inflammatory coupling, tumorous vascular activation, and off-target circulation. Here, the concept that a structure can guide desirable biological function is applied to physically engineer three-dimensional channel networks in implant sites, without any therapeutic treatment. Microchannel networks are generated in a gelatin hydrogel to overcome the diffusion limit of nutrients and oxygen three-dimensionally. Hydrogel implantation in mouse and porcine models of hindlimb ischemia rescues severely damaged tissues by the ingrowth of neighboring host vessels with microchannel perfusion. This effect is guided by microchannel size-specific regenerative macrophage polarization with the consequent functional recovery of endothelial cells. Multiple-site implantation reveals hypoxia and neighboring vessels as major causative factors of the beneficial function. This technique may contribute to the development of therapeutics for hypoxia/inflammatory-related diseases.

Low-Grade Inflammation in Hemophilia

We previously showed that impaired clotting in hemophilia leads to a deficit in macrophage differentiation, which negatively affects critical regenerative macrophage functions such as clot infiltration and red blood cell phagocytosis. These data provide a functional basis for the delayed wound healing as well as protracted joint inflammation commonly observed in hemophiliacs and suggest that altered macrophage function is linked to the activation of the innate immune system. We, therefore, hypothesize that hemophiliacs suffer from chronic low-grade inflammation, which in turn can affect joint health, tissue regeneration and age-related ailments such as cardiovascular disease. For this study, we collected citrated blood from 48 adult male patients with hemophilia A or B with an average age of 36 years and a body mass index (BMI) of 27.7 kg/m2. The majority of patients had a residual FVIII/FIX activity < 1% (77%) and received prophylactic treatment (60%) with a recombinant or plasmatic coagulation factor concentrate. Approximately one-half of the patients had target joints or other bleeding events in the last 3 months and one-third of the patients had contracted HBV, HCV or HIV. For controls, we randomly recruited male blood donors (n = 60; age, 35.8 years; BMI, 27.0) from our blood donation center. To assess inflammation in hemophiliacs, we analyzed platelet-poor plasma from our main collective and a BMI-adjusted cohort using commercially available ELISA kits. The results showed a significant increase of two acute-phase proteins, C-reactive protein and leptin in hemophilia patients compared to healthy controls. Further analysis demonstrated that C-reactive protein and leptin expression inversely correlated with the residual clotting activity as both parameters were high in patients with severe Hemophilia A or B and comparatively low in patients with moderate to mild hemophilia. Of note, there was neither an increase of C-reactive protein or leptin in hemophilia patients with recent bleeding (< 3 month), arthropathy, chronic viral infection nor a decrease in patients with coagulation factor activity > 10% due to prophylactic treatment or recent replacement. Therefore, these data suggest a basic link between clotting deficiencies and chronic low-grade inflammation. Low-grade inflammation is maintained by adipokines, which originate from the adipose tissue and are modulated by a process known as adipose tissue inflammation. In addition to the upregulation of the pro-inflammatory leptin, we detected a significant down-regulation of the anti-inflammatory adiponectin in the plasma of hemophilia patients resulting in a markedly decreased adiponectin/leptin ratio. To enquire if the adipose tissue inflammation in hemophilia originates from gram-negative gut bacteria that translocate into the blood circulation, we also detected elevated plasma levels of lipopolysaccharide-binding protein and hepcidin in hemophilia patients. Together, these data support the concept that low-grade inflammation in hemophilia originates from lipopolysaccharide, which in turn causes adipose tissue inflammation. To test the hypothesis that low-grade inflammation in hemophilia is caused by decreased clotting activity, we collected blood from hemophilia B patients before and after transition from a conventional standard-half-life factor IX concentrate to a prophylactic therapy with an elongated half-life (EHL) FIX (Albutrepennonacog alfa, Idelvion®). Following up on the enhanced factor replacement after > 6 months, we observed a return of hepcidin plasma levels back to baseline values in healthy controls. The decreased hepcidin values from EHL FXI therapy correlated with healing of target joints suggesting that EHL FIX not only controls bleeding but also inflammation. Together, our data demonstrate a specific link between hemophilia and low-grade inflammation that appears to involve increased lipopolysaccharide levels in the blood circulation and subsequent adipose tissue inflammation. In addition, we present evidence that low-grade inflammation is the result of the underlying clotting deficit and that sustained normalization of the clotting deficit with EHL factors ameliorates inflammation. Disclosures Eichler: Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Pilch:CSL Behring: Other: Grants (investigator initiated), Speakers Bureau; ASPIRE Award/Pfizer: Other: Grants (investigator initiated); Bayer: Consultancy, Speakers Bureau; Roche: Consultancy.

Macrophage Polarization Is Impaired in Hemophilia

Macrophages are master regulators of inflammation and wound healing. As such they play an important role in hemophilia, which is commonly associated with delayed tissue regeneration and bleeding-induced joint inflammation. The objective of this study is to determine if macrophage function is deregulated in hemophilia and whether this affects the physiological balance of tissue regeneration and inflammation.For this study, we analyzed monocytes and plasma from a cohort of 26 adult patients with hemophilia A or B that visited our clinic for their annual routine check-up. Patients with acute bleeding events were excluded. The majority of patients had severe forms of hemophilia with factor VIII or IX activity < 1% and, therefore, received prophylactic factor replacement therapy (recombinant or plasmatic) but we also included patients with moderate to mild hemophilia, which were only treated in case of bleeding. Corresponding control samples stemmed from healthy male individuals that we recruited randomly from our blood donor center.To assess macrophage differentiation, we isolated monocytes from the peripheral blood of hemophilia patients as well as healthy controls and began treatment with M-CSF or GM-CSF for 7 days. Macrophage differentiation was confirmed by phase contrast and fluorescence microscopy, which revealed a spread and elongated cell phenotype in donor monocytes in the presence of M-CSF and, to a lesser extent, GM-CSF. Hemophilia monocytes, on the other hand, failed to spread properly in response to either of the cytokines suggesting that macrophage polarization is diminished in patients with reduced activity of coagulation factor VIII or IX.As hemophilia macrophages failed to spread in response to M-CSF, they also failed to express TNFα and CD163, which are macrophage differentiation markers typically induced by M-CSF. In contrast, GM-CSF-induced differentiation was only mildly suppressed suggesting that hemophilia macrophages specifically resist the differentiating stimulus of M-CSF. Consequently, we experienced a significant impairment in M-CSF-induced regenerative macrophage functions such as clot infiltration and red blood cell phagocytosis in hemophilia. Intriguingly, while monocyte invasion was impaired, protein expression in response to M-CSF was regained with respect to CD163 and CD206 after embedding hemophilia monocytes in clotted plasma from healthy blood donors suggesting that a functioning coagulation system has positive effects on regenerative macrophage functions.The inability of hemophilia macrophages to promote M-CSF-mediated signals correlates with a marked down regulation of the M-CSF receptor CSF-1R on hemophilia monocytes as determined in citrated whole blood by flow cytometry. Alongside with a modest reduction of GM-CSF-R, we also detected a substantial reduction of CD163 and Tie2, which are specifically expressed on regenerative monocytes/macrophages, suggesting that clotting deficiencies impair the immune function either directly or indirectly. To further analyze the immune status of hemophilia patients, we performed a cytokine array on plasma samples from hemophiliacs versus healthy blood donors, which revealed the down regulation of a large spectrum of anti-inflammatory and regenerative cytokines in the blood of hemophilia patients.Among the few cytokines upregulated in the blood of hemophiliacs, the adipokine leptin was the most prominent (6-fold, hemophiliac vs. donor). Since leptin has been shown to cause deregulation of the innate immunity, we treated the human monocyte cell line THP1 with recombinant leptin and found a significant inhibitory effect of M-CSF-induced spreading and clot invasion. Therefore, these data suggest that high leptin levels in the blood can reiterate the changes in monocyte function we observed in hemophilia. Together, we conclude that macrophage differentiation is deregulated in hemophilia as a result of resistance towards the cytokine M-CSF. Consequently, hemophilia macrophages are unable to properly perform regenerative functions such as clot invasion and red blood cell phagocytosis. The hemophilic monocyte/macrophage phenotype we describe can be induced by high levels of leptin and mitigated by correcting the clotting dysfunction suggesting a two prone approach to prevent delayed wound healing and persistent inflammation in hemophilia. DisclosuresNo relevant conflicts of interest to declare.

Adipose-Derived Stem Cells in a Resilient In Situ Forming Hydrogel Modulate Macrophage Phenotype.

Injectable hydrogels have the potential to enhance stem cell-based therapies by improving cell localization, retention, and survival after transplantation. The inflammatory response to both the hydrogel and the encapsulated cells is a critical aspect of this strategy, with macrophages being highly involved in the process of hydrogel remodeling, angiogenesis, and tissue regeneration. As a step toward the development of a cell-based strategy for therapeutic angiogenesis, this work compared the intramuscular injection of allogeneic rat adipose-derived stem/stromal cells (rASCs) in an in situ gelling hydrogel with the injection of the hydrogel alone and rASCs in saline in an immunocompetent rat model by immunohistochemical analysis over 4 weeks. rASCs delivered in the hydrogel were retained intramuscularly at significantly higher densities as compared with cells delivered in saline. The encapsulated rASCs modulated the inflammatory response, promoting CD68+ macrophage recruitment, with the majority of infiltrating cells expressing the M1 marker CCR7, as well as a higher fraction of CD163+ M2c macrophages surrounding the hydrogel. Furthermore, rASCs reduced the initial expression of inducible nitric oxide synthase and promoted arginase-1 expression in the infiltrating macrophages over time, consistent with a shift toward a more proregenerative phenotype. Coincident with the enhanced macrophage infiltration, significantly more CD31+ lumens were observed surrounding and within the hydrogels with rASCs at 2 and 4 weeks as compared with the hydrogels alone. Overall, these results are a promising indication that encapsulated rASCs can have immunomodulatory effects and may enhance angiogenic processes after intramuscular injection, promoting a regenerative macrophage response and blood vessel formation.