Macrophage-associated Diseases Research Articles

Macrophage Perspectives in Liver Diseases: Programmed Death, Related Biomarkers, and Targeted Therapy.

Macrophages, as important immune cells of the organism, are involved in maintaining intrahepatic microenvironmental homeostasis and can undergo rapid phenotypic changes in the injured or recovering liver. In recent years, the crucial role of macrophage-programmed cell death in the development and regression of liver diseases has become a research hotspot. Moreover, macrophage-targeted therapeutic strategies are emerging in both preclinical and clinical studies. Given the macrophages' vital role in complex organismal environments, there is tremendous academic interest in developing novel therapeutic strategies that target these cells. This review provides an overview of the characteristics and interactions between macrophage polarization, programmed cell death, related biomarkers, and macrophage-targeted therapies. It aims to deepen the understanding of macrophage immunomodulation and molecular mechanisms and to provide a basis for the treatment of macrophage-associated liver diseases.

α-Aminobutyric Acid Constrains Macrophage-Associated Inflammatory Diseases through Metabolic Reprogramming and Epigenetic Modification.

Metabolites play critical roles in macrophage polarization and in their function in response to infection and inflammation. α-aminobutyric acid (AABA), a non-proteinogenic amino acid which can be generated from methionine, threonine, serine, and glycine, has not been studied extensively in relation to macrophage polarization and function. In this study, we aimed to investigate the immunomodulatory function of AABA in regulating M1 macrophage polarization and function in vitro and in vivo. We stimulated bone-marrow-derived macrophages with lipopolysaccharide (LPS) to generate M1 macrophages. Subsequently, we induced sepsis and colitis in mice, followed by treatment with AABA. We then analyzed the samples using ELISA, real-time PCR, Western blotting, flow cytometry, and histopathological analysis to evaluate cytokine secretion, inflammatory gene expression, macrophage activation, disease progression, and inflammation severity. Additionally, metabolomic and chromatin immunoprecipitation-qPCR were conducted to investigate the function of AABA on metabolic reprogramming and epigenetic modifications of M1 macrophages. Our results revealed that AABA inhibited M1 macrophage polarization and function, which led to prolonged survival in septic mice and reduced disease severity in colitis mice. Mechanically, AABA promoted oxidative phosphorylation (OXPHOS) and glutamine and arginine metabolism while inhibiting glycolysis. Moreover, AABA could increase the occupancy of trimethylation of histone H3K27 at the promoter regions of M1 macrophage-associated inflammatory genes, which contributed to the inhibition of M1 macrophage polarization. These findings suggest that AABA may have therapeutic potential for inflammatory diseases by regulating macrophage polarization and function through metabolic and epigenetic pathways.

Intestinal microbe-dependent \u03c93 lipid metabolite \u03b1KetoA prevents inflammatory diseases in mice and cynomolgus macaques

Dietary ω3 fatty acids have important health benefits and exert their potent bioactivity through conversion to lipid mediators. Here, we demonstrate that microbiota play an essential role in the body’s use of dietary lipids for the control of inflammatory diseases. We found that amounts of 10-hydroxy-cis-12-cis-15-octadecadienoic acid (αHYA) and 10-oxo-cis-12-cis-15-octadecadienoic acid (αKetoA) increased in the feces and serum of specific-pathogen-free, but not germ-free, mice when they were maintained on a linseed oil diet, which is high in α-linolenic acid. Intake of αKetoA, but not αHYA, exerted anti-inflammatory properties through a peroxisome proliferator-activated receptor (PPAR)γ-dependent pathway and ameliorated hapten-induced contact hypersensitivity by inhibiting the development of inducible skin-associated lymphoid tissue through suppression of chemokine secretion from macrophages and inhibition of NF-κB activation in mice and cynomolgus macaques. Administering αKetoA also improved diabetic glucose intolerance by inhibiting adipose tissue inflammation and fibrosis through decreased macrophage infiltration in adipose tissues and altering macrophage M1/M2 polarization in mice fed a high-fat diet. These results collectively indicate that αKetoA is a novel postbiotic derived from α-linolenic acid, which controls macrophage-associated inflammatory diseases and may have potential for developing therapeutic drugs as well as probiotic food products.

Metabolite transporters as regulators of macrophage polarization.

Macrophages are myeloid immune cells, present in virtually all tissues which exhibit considerable functional plasticity and diversity. Macrophages are often subdivided into two distinct subsets described as classically activated (M1) and alternatively activated (M2) macrophages. It has recently emerged that metabolites regulate the polarization and function of macrophages by altering metabolic pathways. These metabolites often cannot freely pass the cell membrane and are therefore transported by the corresponding metabolite transporters. Here, we reviewed how glucose, glutamate, lactate, fatty acid, and amino acid transporters are involved in the regulation of macrophage polarization. Understanding the interactions among metabolites, metabolite transporters, and macrophage function under physiological and pathological conditions may provide further insights for novel drug targets for the treatment of macrophage-associated diseases. In Brief Recent studies have shown that the polarization and function of macrophages are regulated by metabolites, most of which cannot pass freely through biofilms. Therefore, metabolite transporters required for the uptake of metabolites have emerged seen as important regulators of macrophage polarization and may represent novel drug targets for the treatment of macrophage-associated diseases. Here, we summarize the role of metabolite transporters as regulators of macrophage polarization.

Selective Induction of Cell Death in Human M1 Macrophages by Smac Mimetics Is Mediated by cIAP-2 and RIPK-1/3 through the Activation of mTORC.

Inflammatory macrophages have been implicated in many diseases, including rheumatoid arthritis and inflammatory bowel disease. Therefore, targeting macrophage function and activation may represent a potential strategy to treat macrophage-associated diseases. We have previously shown that IFN-γ-induced differentiation of human M0 macrophages toward proinflammatory M1 state rendered them highly susceptible to the cytocidal effects of second mitochondria-derived activator of caspases mimetics (SMs), antagonist of the inhibitors of apoptosis proteins (IAPs), whereas M0 and anti-inflammatory M2c macrophages were resistant. In this study, we investigated the mechanism governing SM-induced cell death during differentiation into M1 macrophages and in polarized M1 macrophages. IFN-γ stimulation conferred on M0 macrophages the sensitivity to SM-induced cell death through the Jak/STAT, IFN regulatory factor-1, and mammalian target of rapamycin complex-1 (mTORC-1)/ribosomal protein S6 kinase pathways. Interestingly, mTORC-1 regulated SM-induced cell death independent of M1 differentiation. In contrast, SM-induced cell death in polarized M1 macrophages is regulated by the mTORC-2 pathway. Moreover, SM-induced cell death is regulated by cellular IAP (cIAP)-2, receptor-interacting protein kinase (RIPK)-1, and RIPK-3 degradation through mTORC activation during differentiation into M1 macrophages and in polarized M1 macrophages. In contrast to cancer cell lines, SM-induced cell death in M1 macrophages is independent of endogenously produced TNF-α, as well as the NF-κB pathway. Collectively, selective induction of cell death in human M1 macrophages by SMs may be mediated by cIAP-2, RIPK-1, and RIPK-3 degradation through mTORC activation. Moreover, blocking cIAP-1/2, mTORC, or IFN regulatory factor-1 may represent a promising therapeutic strategy to control M1-associated diseases.

Glycinergic Signaling in Macrophages and Its Application in Macrophage-Associated Diseases.

Accumulating evidences support that amino acids direct the fate decision of immune cells. Glycine is a simple structural amino acid acting as an inhibitory neurotransmitter. Besides, glycine receptors as well as glycine transporters are found in macrophages, indicating that glycine alters the functions of macrophages besides as an inhibitory neurotransmitter. Mechanistically, glycine shapes macrophage polarization via cellular signaling pathways (e.g., NF-κB, NRF2, and Akt) and microRNAs. Moreover, glycine has beneficial effects in preventing and/or treating macrophage-associated diseases such as colitis, NAFLD and ischemia-reperfusion injury. Collectively, this review highlights the conceivable role of glycinergic signaling for macrophage polarization and indicates the potential application of glycine supplementation as an adjuvant therapy in macrophage-associated diseases.

Ironing Out the Details: How Iron Orchestrates Macrophage Polarization.

Iron fine-tunes innate immune responses, including macrophage inflammation. In this review, we summarize the current understanding about the iron in dictating macrophage polarization. Mechanistically, iron orchestrates macrophage polarization through several aspects, including cellular signaling, cellular metabolism, and epigenetic regulation. Therefore, iron modulates the development and progression of multiple macrophage-associated diseases, such as cancer, atherosclerosis, and liver diseases. Collectively, this review highlights the crucial role of iron for macrophage polarization, and indicates the potential application of iron supplementation as an adjuvant therapy in different inflammatory disorders relative to the balance of macrophage polarization.

Renin\u2013angiotensin system impairs macrophage lipid metabolism to promote age-related macular degeneration in mouse models

Metabolic syndrome, a condition involving obesity and hypertension, increases the risk of aging-associated diseases such as age-related macular degeneration (AMD). Here, we demonstrated that high-fat diet (HFD)-fed mice accumulated oxidized low-density lipoprotein (ox-LDL) in macrophages through the renin–angiotensin system (RAS). The ox-LDL-loaded macrophages were responsible for visual impairment in HFD mice along with a disorder of the retinal pigment epithelium (RPE), which is required for photoreceptor outer segment renewal. RAS repressed ELAVL1, which reduced PPARγ, impeding ABCA1 induction to levels that are sufficient to excrete overloaded cholesterol within the macrophages. The ox-LDL-loaded macrophages expressed inflammatory cytokines and attacked the RPE. An antihypertensive drug, angiotensin II type 1 receptor (AT1R) blocker, resolved the decompensation of lipid metabolism in the macrophages and reversed the RPE condition and visual function in HFD mice. AT1R signaling could be a future therapeutic target for macrophage-associated aging diseases, such as AMD.

Abstract B50: Tissue factor-targeting CAR-NK cells for immunotherapy of triple-negative breast cancer

Abstract The objective of the study is to develop tissue factor (TF)-targeted chimeric antigen receptor-natural killer (TF-CAR-NK) cell immunotherapy for the treatment of triple-negative breast cancer (TNBC). TNBC represents nearly 15% of globally diagnosed breast cancer, which is also the most frequently diagnosed cancer in women in the United States. Due to the lack of validated therapeutic target molecules, TNBC is one of the most difficult-to-treat malignancies, and, in most cases, is considered an incurable malignancy. CAR-expressing NK and T-cell therapy represents a novel cancer immunotherapy approach. To address the unmet issue, we first identified that TF is a novel, common, yet selective membrane-bound surface protein receptor and oncotarget for TNBC on the TNBC cancer cells, tumor neovascular endothelial cells and cancer stem cells. It is worth noting that under physiologic conditions, TF is not expressed on normal peripheral lymphocytes such as NK, B, T cells and monocytes, nor on the cells (quiescent vascular endothelial cells) forming the inner layer of normal vasculature, but is solely restricted on some cells (such as pericytes and smooth muscle cells) that are located outside the blood circulation and inner layer of blood vessel walls. Then we developed and tested a novel TF-targeting CAR-NK cell line (NK92MI, a human NK line derived from NK92 after being transfected for endogenous production of interleukin-2) expressing factor VII light chain, natural ligand for TF, as TF-targeting domain followed by CD28-4-1BB-CD3zeta with or without a hinge region of human IgG1 as TF-CAR1 dimer or monomer for CAR-NK immunotherapy of TNBC. Our preclinical results demonstrate that both the TF-CAR1 dimer and monomer-NK cells can kill TNBC cells in vitro and are effective and safe for the treatment of TNBC in mouse models of TNBC cell line-derived and patient’s tumor-derived xenografts (CDX and PDX). Thus, this study established the proof of concept of targeting TF for CAR-NK immunotherapy of TNBC and may warrant further investigation for TNBC patients. Accumulating evidence suggested that TF is selectively expressed in pathologic angiogenesis-dependent as well as macrophage-associated human diseases. Pathologic angiogenesis, the formation of neovasculature, is involved in many clinically significant human diseases, notably cancer (including solid cancer, acute leukemia and sarcoma), age-related macular degeneration, endometriosis and rheumatoid arthritis, whereas macrophage is involved in the progression of a variety of human diseases, such as atherosclerosis and viral infections (HIV and Ebola). If successful, TF-CAR immunotherapy may have potential to treat a broad range of angiogenesis-dependent or macrophage-associated human diseases. Acknowledgments: This work was supported by the Dr. Ralph and Marian Falk Medical Research Trust Awards Programs and partly by an NCI grant (R21CA216697). Z.H. is the inventor of PCT/U.S. Pending Patents on “Tissue Factor-Targeting CAR-NK and CAR-T Cell Therapy.” Citation Format: Zhiwei Hu. Tissue factor-targeting CAR-NK cells for immunotherapy of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B50.

Activated cholangiocytes release macrophage-polarizing extracellular vesicles bearing the DAMP S100A11.

In mouse models of biliary tract diseases, macrophages are recruited to the periductal milieu and promote injury and cholestasis. Although cell necrosis with release of biomolecules termed damage-associated molecular patterns (DAMPs) promotes recruitment and activation of macrophages, necrosis was not observed in these studies. Because extracellular vesicles (EVs) are important in cell-to-cell communication, we postulated that activated cholangiocytes may release EVs containing DAMPs as cargo. Both the human (NHC) and mouse cholangiocyte (603B) cell lines display constitutive activation with mRNA expression of chemokines. Proteomic analysis revealed that EVs from both cell lines contained the DAMP S100A11, a ligand for the receptor for advanced glycation end products (RAGE). Bone marrow-derived macrophages (BMDM) incubated with EVs derived from the mouse 603B cell line increased mRNA expression of proinflammatory cytokines. Genetic or pharmacologic inhibition of RAGE reduced BMDM expression of proinflammatory cytokines treated with EVs. RAGE signaling resulted in activation of the canonical NF-κB pathway, and consistently, proinflammatory cytokine expression was blunted by the IKKα/β inhibitor TPCA-1 in BMDM incubated with EVs. We also demonstrated that primary mouse cholangiocyte-derived organoids express chemokines indicating cholangiocyte activation, release EVs containing S100A11, and stimulate proinflammatory cytokine expression in BMDM by a RAGE-dependent pathway. In conclusion, these observations identify a non-cell death mechanism for cellular release of DAMPs by activated cholangiocytes, namely by releasing DAMPs as EV cargo. These data also suggest RAGE inhibitors may be salutary in macrophage-associated inflammatory diseases of the bile ducts.

Melatonin in macrophage biology: Current understanding and future perspectives.

Melatonin is a ubiquitous hormone found in various organisms and highly affects the function of immune cells. In this review, we summarize the current understanding of the significance of melatonin in macrophage biology and the beneficial effects of melatonin in macrophage-associated diseases. Enzymes associated with synthesis of melatonin, as well as membrane receptors for melatonin, are found in macrophages. Indeed, melatonin influences the phenotype polarization of macrophages. Mechanistically, the roles of melatonin in macrophages are related to several cellular signaling pathways, such as NF-κB, STATs, and NLRP3/caspase-1. Notably, miRNAs (eg, miR-155/-34a/-23a), cellular metabolic pathways (eg, α-KG, HIF-1α, and ROS), and mitochondrial dynamics and mitophagy are also involved. Thus, melatonin modulates the development and progression of various macrophage-associated diseases, such as cancer and rheumatoid arthritis. This review provides a better understanding about the importance of melatonin in macrophage biology and macrophage-associated diseases.

Enhancement of macrophage uptake via phosphatidylserine-coated acetalated dextran nanoparticles

Although vital to the immune system, macrophages can act as reservoirs for pathogens such as tuberculosis and human immunodeficiency virus. Limitations in the treatment of such diseases include targeting therapeutics directly to macrophages and the large systemic dosages needed. The objective of this study is to develop a nanoparticle (NP)-based drug delivery system that can provide targeted delivery into macrophages. Acetalated dextran (Ac-Dex) NP loaded with the lipophilic model compound curcumin (CUR) were synthesized and coated in 1,2-dipalmitoyl-sn-glycero-3-phospho-l-serine (DPPS), a phospholipid that induces phagocytosis in macrophages. DPPS-CUR NP were found to release 67.8% of encapsulated CUR within 24 h at pH 5.35 and exhibited minimal CUR release (6.3%) at pH 7.4. DPPS-CUR NP were uptaken by murine macrophages significantly more than NP without DPPS coating and NP exposure to these macrophages resulted in minimal toxicity to the cells and minimal nitric oxide production. These results suggest that the combination of the DPPS coating and pH-sensitive polymer Ac-Dex can provide a NP delivery system capable of enhanced uptake by macrophages and potential systemic stability to more effectively deliver drugs of interest. As a result, the described DPPS-CUR NP can serve as a viable delivery system for the treatment of macrophage-associated diseases.

The potential of xanthones as a therapeutic option in macrophage-associated inflammatory diseases

Xanthones are well known for their significant biological activities and can be found in many herbal medicines. These compounds have the ability to regulate various inflammatory activities and signaling pathways in immune cells, especially macrophages. Macrophages are innate immune cells that can either fuel or dampen an inflammatory response depending on their activation states and play an active role in the development of inflammatory diseases such as atherosclerosis, arthritis, cancer, and diabetes. Many traditional medicines used as a remedy for these diseases contain xanthones, and their bioactivities may be partially attributed to their ability in regulating macrophage responses. In this review, we discuss the in vitro and in vivo findings on the effects of xanthones on different macrophage immune functions including nitric oxide and cytokine production, migration, polarization, and phagocytosis. Their specific modes of action are highlighted whenever known. We also discuss the potential and challenges in using xanthones as a therapeutic option in various inflammatory diseases. It is hoped that this review can pave the way for future research that focuses on developing xanthones as specific macrophage-targeted therapeutics.

CD100 Effects in Macrophages and Its Roles in Atherosclerosis.

CD100 or Sema4D is a protein from the semaphorin family with important roles in the vascular, nervous and immune systems. It may be found as a membrane bound dimer or as a soluble molecule originated by proteolytic cleavage. Produced by the majority of hematopoietic cells including B and T lymphocytes, natural killer and myeloid cells, as well as endothelial cells, CD100 exerts its actions by binding to different receptors depending on the cell type and on the organism. Cell-to-cell adhesion, angiogenesis, phagocytosis, T cell priming, and antibody production are examples of the many functions of this molecule. Of note, high CD100 serum levels has been found in inflammatory as well as in infectious diseases, but the roles of the protein in the pathogenesis of these diseases has still to be clarified. Macrophages are highly heterogeneous cells present in almost all tissues, which may change their functions in response to microenvironmental conditions. They are key players in the innate and adaptive immune responses and have decisive roles in sterile conditions but also in several diseases such as atherosclerosis, autoimmunity, tumorigenesis, and antitumor responses, among others. Although it is known that macrophages express CD100 and its receptors, few studies have focused on the role of this semaphorin in this cell type or in macrophage-associated diseases. The aim of this review is to critically revise the available data about CD100 and atherosclerosis, with special emphasis on its roles in macrophages and monocytes. We will also describe the few available data on treatments with anti-CD100 antibodies in different diseases. We hope that this review stimulates future studies on the effects of such an important molecule in a cell type with decisive roles in inflammatory diseases such as atherosclerosis.

Synthetic polymannose as a drug carrier: synthesis, toxicity and anti-fungal activity of polymannose-amphotericin B conjugates

Polymannose (PM) having a weight-average molar mass (Mw) of 30–53 kDa was synthesized by the polycondensation of mannose using phosphorous acid as the catalyst and characterized by various techniques such as NMR, IR, GPC and polarimetry. 2D NMR results confirmed the presence of (1 → 6)-linked α-D-mannose residues as backbone with O-3 and O-2 substituted linear or branched chains in PM. Amphotericin B (AmB) was conjugated to periodate-oxidized PM through Schiff’s linkages at 20 wt% concentration. The AmB-PM conjugates were highly soluble in phosphate buffered saline (180–250 mg/mL), exhibited negligible hemolytic potential to human erythrocytes even at a concentration of 200 μg/mL (equivalent to ~40 μg/mL AmB) and were non-toxic to human embryonic kidney (HEK293T) cells even at a concentration of 250 μg/mL (equivalent to ~50 μg/mL AmB). The minimum inhibitory concentration of the AmB-PM conjugates against C. albicans, C. parapsilosis and C. neoformans was in the range of 0.5–1.0 μg/mL. Mannose receptors are widely expressed on myeloid cells such as macrophages, neutrophils, and dendritic cells. Therefore, apart from treating fungal infections, AmB-PM conjugates also may have therapeutic potential for the treatment of macrophage-associated diseases such as leishmaniasis where mannose receptors are overexpressed.

Recent advances in understanding Leishmania donovani infection: The importance of diverse host regulatory pathways

Invasion of host cell by pathogens induce various intracellular signalling pathways. The host cell through the initiation of these signalling circuits desperately wants to get rid of the pathogen, whereas the pathogen tries to subvert these defence strategies to create an environment for their successful survival. Leishmania spp. is not an exception. Leishmania have to evolve a range of strategic mechanisms to neutralize macrophage defensive arsenals which enable the parasite to replicate within the phagolysosome of infected host. Understanding these signalling mechanisms in detail will not only improve our basic knowledge of host-pathogen interaction but will also help us to develop effective drug targets not only against leishmaniasis but also for many other macrophage associated diseases. © 2018 IUBMB Life, 70(7):593-601, 2018.

Therapeutic Antibody-Like Immunoconjugates against Tissue Factor with the Potential to Treat Angiogenesis-Dependent as Well as Macrophage-Associated Human Diseases.

Accumulating evidence suggests that tissue factor (TF) is selectively expressed in pathological angiogenesis-dependent as well as macrophage-associated human diseases. Pathological angiogenesis, the formation of neovasculature, is involved in many clinically significant human diseases, notably cancer, age-related macular degeneration (AMD), endometriosis and rheumatoid arthritis (RA). Macrophage is involved in the progression of a variety of human diseases, such as atherosclerosis and viral infections (human immunodeficiency virus, HIV and Ebola). It is well documented that TF is selectively expressed on angiogenic vascular endothelial cells (VECs) in these pathological angiogenesis-dependent human diseases and on disease-associated macrophages. Under physiology condition, TF is not expressed by quiescent VECs and monocytes but is solely restricted on some cells (such as pericytes) that are located outside of blood circulation and the inner layer of blood vessel walls. Here, we summarize TF expression on angiogenic VECs, macrophages and other diseased cell types in these human diseases. In cancer, for example, the cancer cells also overexpress TF in solid cancers and leukemia. Moreover, our group recently reported that TF is also expressed by cancer-initiating stem cells (CSCs) and can serve as a novel oncotarget for eradication of CSCs without drug resistance. Furthermore, we review and discuss two generations of TF-targeting therapeutic antibody-like immunoconjugates (ICON and L-ICON1) and antibody-drug conjugates that are currently being tested in preclinical and clinical studies for the treatment of some of these human diseases. If efficacy and safety are proven in current and future clinical trials, TF-targeting immunoconjugates may provide novel therapeutic approaches with potential to broadly impact the treatment regimen of these significant angiogenesis-dependent, as well as macrophage-associated, human diseases.

Histone deacetylase 6 modulates macrophage infiltration during inflammation.

Mice with histone deacetylase 6 (HDAC6) deficiency grow and develop normally but exhibit impaired immune response. The molecular mechanisms for this phenotype remain largely elusive.Methods: A mouse acute peritonitis model was used to study the infiltration of neutrophils and monocyte-derived macrophages. In vitro cell motility assays were performed to analyze monocyte/macrophage recruitment. Fluorescence microscopy and flow cytometry were performed to examine the phagocytic ability of macrophages. Immunofluorescence microscopy was used to investigate protein localization, protrusion formation, and microtubule acetylation.Results: HDAC6 deficiency does not affect neutrophil infiltration, but instead attenuates the infiltration of monocyte-derived macrophages into the peritoneal cavity. HDAC6 plays a specific role in monocyte/macrophage recruitment. Loss of HDAC6 suppresses the phagocytic capacity of macrophages challenged with E. coli. Lipopolysaccharide stimulation results in the translocation of HDAC6 and cortactin from the cytosol to the cell periphery, promotes the formation of filopodial protrusions, and enhances microtubule acetylation around the microtubule-organizing center, all of which are abrogated by HDAC6 deficiency.Conclusion: These findings implicate HDAC6 in the innate immune response and suggest that it may serve as a promising target for the treatment of macrophage-associated immune diseases.

Thermoresponsive hyaluronic acid nanogels as hydrophobic drug carrier to macrophages

Delivery systems for macrophages are particularly attractive since these phagocytic cells play a important role in immunological and inflammatory responses, also acting as host cells for microorganisms that are involved in deadly infectious diseases, such as leishmaniasis. Hyaluronic acid (HA) is specifically recognized by macrophages that are known to express HA receptors. Therefore, in this study, we focused on HA-based nanogels as drug carriers for these cells. The drug delivery was validated in an in vivo study on mice using intravital two-photon laser scanning microscopy. HA derivatives were modified with a biocompatible oligo(ethylene glycol)-based thermoresponsive polymer to form nanogels. These HA conjugates were readily prepared by varying the molar mass of initial HA and the degree of substitution via radical-mediated thiol-ene chemistry in aqueous solution. The derivatives were shown to self-assemble into spherical gel particles with diameters ranging from 150 to 214nm above 37°C. A poorly water-soluble two-photon dye was successfully loaded into the nanogels during this self-assembly process. In vitro cellular uptake tests using a RAW 264.7 murine macrophage cell line showed successful intracellular delivery of the hydrophobic dye. After intravenous injection in mice, the nanogels circulated freely in the blood but were rapidly phagocytized within 13min by circulating macrophages and stored in the liver and spleen, as observed by two-photon microscopy. Benefit can be thus expected in using such a delivery system for the liver and spleen macrophage-associated diseases.

In vivo detection of macrophage recruitment in hind-limb ischemia using a targeted near-infrared fluorophore.

Macrophages are an essential component of the immune system and have protective and pathogenic functions in various diseases. Imaging of macrophages in vivo could furnish new tools to advance evaluation of disease and therapies. Critical limb ischemia is a disease in which macrophages have considerable pathogenic roles, and are potential targets for cell-based immunotherapy. We sought to develop a new near-infrared fluorescence (NIRF) imaging probe to target macrophages specifically in vivo in various pathological states, including hind-limb ischemia. We rapidly screened the photostable cyanine-based NIRF library against different blood cell lines. The identified monocyte/macrophage-selective hit was tested in vitro in live-cell labeling assay. Non-invasive NIRF imaging was performed with murine models of paw inflammation by lipopolysaccharide challenge and hind-limb ischemia with femoral artery ligation. in vivo macrophage targeting was further evaluated using intravital microscopy with Csf1r-EGFP transgenic mice and immunofluorescent staining with macrophage-specific markers. We discovered MF800, a Macrophage-specific near-infrared Fluorophore, which showed selective live-cell imaging performance in a panel of cell lines and primary human blood samples. MF800 outperforms the clinically-available NIRF contrast agent ICG for in vivo specificity in paw inflammation and hind-limb ischemia models. We observed a marked overlap of MF800-labeled cells and EGFP-expressing macrophages in intravital imaging of Csf1r-EGFP transgenic mice. In the histologic analysis, MF800-positive cells also expressed the macrophage markers CD68 and CD169. NIRF imaging showcased the potential of using MF800 to understand macrophage behavior in vivo, characterize macrophage-associated diseases, and may help in assessing therapeutic responses in the clinic.