Novel modification of Luminex assay for characterization of extracellular vesicle populations in biofluids.

Most approaches to extracellular vesicle (EV) characterization focus on EV size or density. However, such approaches provide few clues regarding EV origin, molecular composition, and function. New methods to characterize the EV surface proteins may aid our understanding of their origin, physiological roles, and biomarker potential. Recently developed immunoassays for intact EVs based on ELISA, NanoView, SIMOA and MesoScale platforms are highly sensitive, but have limited multiplexing capabilities, whereas MACSPlex FACS enables the detection of multiple EV surface proteins, but requires significant quantities of purified EVs, which limits its adoption. Here, we describe a novel Luminex-based immunoassay, which combines multiplexing capabilities with high sensitivity and, importantly, bypasses the enrichment and purification steps that require larger sample volumes. We demonstrate the method specificity for detecting EV surface proteins using multiple EV depletion techniques, EVs of specific cellular origin isolated from culture media, and by co-localization with established EV surface markers. Using this novel approach, we elucidate differences in the tetraspanin profiles of the EVs carrying erythrocyte and neuron markers. Using size exclusion chromatography, we show that plasma EVs of putative neuronal and tissue macrophage origin are eluted in fractions distinct from those derived from erythrocytes, or from their respective cultured cells. In conclusion, our novel multiplexed assay differentiates between EVs from erythrocytes, macrophages, and neurons, and offers a new means for capture, classification, and profiling of EVs from diverse sources.

Radiolabeled Monoclonal Antibody Against Colony-Stimulating Factor 1 Receptor Specifically Distributes to the Spleen and Liver in Immunocompetent Mice

Macrophages can promote tumor development. Preclinically, targeting macrophages by colony-stimulating factor 1 (CSF1)/CSF1 receptor (CSF1R) monoclonal antibodies (mAbs) enhances conventional therapeutics in combination treatments. The physiological distribution and tumor uptake of CSF1R mAbs are unknown. Therefore, we radiolabeled a murine CSF1R mAb and preclinically visualized its biodistribution by PET. CSF1R mAb was conjugated to N-succinyl-desferrioxamine (N-suc-DFO) and subsequently radiolabeled with zirconium-89 (89Zr). Optimal protein antibody dose was first determined in non-tumor-bearing mice to assess physiological distribution. Next, biodistribution of optimal protein dose and 89Zr-labeled isotype control was compared with PET and ex vivo biodistribution after 24 and 72 h in mammary tumor-bearing mice. Tissue autoradiography and immunohistochemistry determined radioactivity distribution and tissue macrophage presence, respectively. [89Zr]Zr-DFO-N-suc-CSF1R-mAb optimal protein dose was 10 mg/kg, with blood pool levels of 10 ± 2% injected dose per gram tissue (ID/g) and spleen and liver uptake of 17 ± 4 and 11 ± 4%ID/g at 72 h. In contrast, 0.4 mg/kg of [89Zr]Zr-DFO-N-suc-CSF1R mAb was eliminated from circulation within 24 h; spleen and liver uptake was 126 ± 44% and 34 ± 7%ID/g, respectively. Tumor-bearing mice showed higher uptake of [89Zr]Zr-DFO-N-suc-CSF1R-mAb in the liver, lymphoid tissues, duodenum, and ileum, but not in the tumor than did 89Zr-labeled control at 72 h. Immunohistochemistry and autoradiography showed that 89Zr was localized to macrophages within lymphoid tissues. Following [89Zr]Zr-DFO-N-suc-CSF1R-mAb administration, tumor macrophages were almost absent, whereas isotype-group tumors contained over 500 cells/mm2. We hypothesize that intratumoral macrophage depletion by [89Zr]Zr-DFO-N-suc-CSF1R-mAb precluded tumor uptake higher than 89Zr-labeled control. Translation of molecular imaging of macrophage-targeting therapeutics to humans may support macrophage-directed therapeutic development.

Metabolism of tissue macrophages in homeostasis and pathology

Cellular metabolism orchestrates the intricate use of tissue fuels for catabolism and anabolism to generate cellular energy and structural components. The emerging field of immunometabolism highlights the importance of cellular metabolism for the maintenance and activities of immune cells. Macrophages are embryo- or adult bone marrow-derived leukocytes that are key for healthy tissue homeostasis but can also contribute to pathologies such as metabolic syndrome, atherosclerosis, fibrosis or cancer. Macrophage metabolism has largely been studied in vitro. However, different organs contain diverse macrophage populations that specialize in distinct and often tissue-specific functions. This context specificity creates diverging metabolic challenges for tissue macrophage populations to fulfill their homeostatic roles in their particular microenvironment and conditions their response in pathological conditions. Here, we outline current knowledge on the metabolic requirements and adaptations of macrophages located in tissues during homeostasis and selected diseases.

Fetuin-A regulates adipose tissue macrophage content and activation in insulin resistant mice through MCP-1 and iNOS: Involvement of IFNγ-JAK2-STAT1 pathway

In the context of obesity-induced adipose tissue inflammation, migration of macrophages and their polarization from predominantly anti-inflammatory to proinflammatory subtype is considered a pivotal event in the loss of adipose insulin sensitivity. Two major chemoattractants, monocyte chemoattractant protein-1 (MCP-1) and Fetuin A (FetA), have been reported to stimulate macrophage migration into inflamed adipose tissue instigating inflammation. Moreover, FetA could notably modulate macrophage polarization, yet the mechanism(s) is unknown. The present study was undertaken to elucidate the mechanistic pathway involved in the actions of FetA and MCP-1 in obese adipose tissue. We found that FetA knockdown in high fat diet (HFD) fed mice could significantly subdue the augmented MCP-1 expression and reduce adipose tissue macrophage (ATM) content thereby indicating that MCP-1 is being regulated by FetA. Additionally, knockdown of FetA in HFD mice impeded the expression of inducible nitric oxide synthase (iNOS) reverting macrophage activation from mostly proinflammatory to anti-inflammatory state. It was observed that the stimulating effect of FetA on MCP-1 and iNOS was mediated through interferon γ (IFNγ) induced activation of JAK2-STAT1-NOX4 pathway. Furthermore, we detected that the enhanced IFNγ expression was accounted by the stimulatory effect of FetA upon the activities of both cJun and JNK. Taken together, our findings revealed that obesity-induced FetA acts as a master upstream regulator of adipose tissue inflammation by regulating MCP-1 and iNOS expression through JNK-cJun-IFNγ-JAK2-STAT1 signaling pathway. This study opened a new horizon in understanding the regulation of ATM content and activation in conditions of obesity-induced insulin resistance.

Chromatin accessibility analysis identifies the transcription factor ETV5 as a suppressor of adipose tissue macrophage activation in obesity

Activation of adipose tissue macrophages (ATMs) contributes to chronic inflammation and insulin resistance in obesity. However, the transcriptional regulatory machinery involved in ATM activation during the development of obesity is not fully understood. Here, we profiled the chromatin accessibility of blood monocytes and ATMs from obese and lean mice using assay for transposase-accessible chromatin sequencing (ATAC-seq). We found that monocytes and ATMs from obese and lean mice exhibited distinct chromatin accessibility status. There are distinct regulatory elements that are specifically associated with monocyte or ATM activation in obesity. We also discovered several transcription factors that may regulate monocyte and ATM activation in obese mice, specifically a predicted transcription factor named ETS translocation variant 5 (ETV5). The expression of ETV5 was significantly decreased in ATMs from obese mice and its downregulation was mediated by palmitate stimulation. The decrease in ETV5 expression resulted in macrophage activation. Our results also indicate that ETV5 suppresses endoplasmic reticulum (ER) stress and Il6 expression in macrophages. Our work delineates the changes in chromatin accessibility in monocytes and ATMs during obesity, and identifies ETV5 as a critical transcription factor suppressing ATM activation, suggesting its potential use as a therapeutic target in obesity-related chronic inflammation.

Preventing ovariectomy-induced weight gain decreases tumor burden in rodent models of obesity and postmenopausal breast cancer.

Obesity and adult weight gain are linked to increased breast cancer risk and poorer clinical outcomes in postmenopausal women, particularly for hormone-dependent tumors. Menopause is a time when significant weight gain occurs in many women, and clinical and preclinical studies have identified menopause (or ovariectomy) as a period of vulnerability for breast cancer development and promotion. We hypothesized that preventing weight gain after ovariectomy (OVX) may be sufficient to prevent the formation of new tumors and decrease growth of existing mammary tumors. Here, we tested this hypothesis in a rat model of obesity and carcinogen-induced postmenopausal mammary cancer and validated our findings in a murine xenograft model with implanted human tumors. In both models, preventing weight gain after OVX significantly decreased obesity-associated tumor development and growth. Importantly, we did not induce weight loss in these animals, but simply prevented weight gain. In both lean and obese rats, preventing weight gain reduced visceral fat accumulation and associated insulin resistance. Similarly, the intervention decreased circulating tumor-promoting growth factors and inflammatory cytokines (ie, BNDF, TNFα, FGF2), with greater effects in obese compared to lean rats. In obese rats, preventing weight gain decreased adipocyte size, adipose tissue macrophage infiltration, reduced expression of the tumor-promoting growth factor FGF-1, and reduced phosphorylated FGFR in tumors. Together, these findings suggest that the underlying mechanisms associated with the anti-tumor effects of weight maintenance are multi-factorial, and that weight maintenance during the peri-/post-menopausal period may be a viable strategy for reducing obesity-associated breast cancer risk and progression in women.

A kinase-dead Csf1r mutation associated with adult-onset leukoencephalopathy has a dominant-negative impact on CSF1R signaling

Amino acid substitutions in the kinase domain of the human CSF1R gene are associated with autosomal dominant adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). To model the human disease, we created a disease-associated mutation (Glu631Lys; E631K) in the mouse Csf1r locus. Homozygous mutation (Csf1rE631K/E631K) phenocopied the Csf1r knockout; with prenatal mortality or severe postnatal growth retardation and hydrocephalus. Heterozygous mutation delayed the postnatal expansion of tissue macrophage populations in most organs. Bone marrow cells from Csf1rE631K/+ mice were resistant to CSF1 stimulation in vitro, and Csf1rE631K/+ mice were unresponsive to administration of a CSF1-Fc fusion protein which expands tissue macrophage populations in controls. In the brain, microglial cell numbers and dendritic arborization were reduced in the Csf1rE631K/+ mice as in ALSP patients. The microglial phenotype is the opposite of microgliosis observed in Csf1r+/- mice. However, we found no evidence of brain pathology or impacts on motor function in aged Csf1rE631K/+ mice. We conclude that disease-associated CSF1R mutations encode dominant negative repressors of CSF1R signaling. We speculate that leukoencephalopathy associated with human CSF1R mutations requires an environmental trigger and/or epistatic interaction with common neurodegenerative disease-associated alleles.Summary StatementThis study describes the effect of a human disease-associated mutation in the mouse CSF1R gene on postnatal development and growth factor responsiveness of cells of the macrophage lineage.

Adipose Tissue Immunometabolism and Apoptotic Cell Clearance

The safe removal of apoptotic debris by macrophages—often referred to as efferocytosis—is crucial for maintaining tissue integrity and preventing self-immunity or tissue damaging inflammation. Macrophages clear tissues of hazardous materials from dying cells and ultimately adopt a pro-resolving activation state. However, adipocyte apoptosis is an inflammation-generating process, and the removal of apoptotic adipocytes by so-called adipose tissue macrophages triggers a sequence of events that lead to meta-inflammation and obesity-associated metabolic diseases. Signals that allow apoptotic cells to control macrophage immune functions are complex and involve metabolites released by the apoptotic cells and also metabolites produced by the macrophages during the digestion of apoptotic cell contents. This review provides a concise summary of the adipocyte-derived metabolites that potentially control adipose tissue macrophage immune functions and, hence, may induce or alleviate adipose tissue inflammation.

Application of Novel Drug Delivery System for Herbal Formulations

Novel Drug Delivery System (NDDS) is defined to the approaches, formulations, technologies, and system which provide a therapeutic amount of drug to the appropriate site in the body. If the novel drug delivery technology is applied in herbal actives and extracts. It may help in increasing the efficacy and reducing the side effect of variety of novel herbal formulation like polymeric nanoparticle, nanocapsules, liposomes, nanoemulsion, phytosomes, microsphere and ethosomes has been reported using bioactive and plant extracts. These novel formulations have advantage over the conventional formulations. NDDS include enhancement of solubility, bioavailability, protection against toxicity, enhancement of pharmacological activity and stability, improve tissue macrophage distribution and protection against chemical degradation. The most important aim to design alternative drug delivery technologies is to increase efficiency and safety of drug in the process of drug delivery and provide more convenience for the patients. The present paper includes more information regarding novel formulation of herbal formulation.