Lysosomes Of Macrophages Research Articles

Low density lipoprotein oxidized under lysosomal conditions decreases arterial vasodilatation

Endothelial dysfunction is a risk factor for atherosclerosis and includes impaired endothelium-dependent vasodilatation. We have shown previously that low density lipoprotein (LDL) can be oxidized by iron in the lysosomes of macrophages. Macrophage lysis in atherosclerotic lesions might expose endothelial cells to this oxidized LDL and adversely affect their function. LDL was oxidized by ferrous sulfate (5 µM) for 24 h at pH 4.5 at 37 °C. Aortas from male Wistar rats were cut into rings and subjected to wire myography for isometric tension recording. The rings were incubated with or without oxidized LDL (50 µg protein/ml) for one hour, constricted with 100 nM phenylephrine and relaxation to acetylcholine (1 nM − 3 µM) was measured. There was about 50% less relaxation in the presence of this oxidized LDL. Endothelial-independent vasodilatation induced by sodium nitroprusside was less affected by oxidized LDL. Oxidized LDL increased the formation of reactive oxygen species by the aortic rings and by cultured human aortic and dermal microvascular endothelial cells, which might have inactivated nitric oxide. Acetylcholine increased the activatory phosphorylation of eNOS (ser-1177), but oxidized LDL had little effect on this activation in cultured human aortic endothelial cells. These findings raise the possibility that LDL oxidized in lysosomes and released from lysed macrophages might decrease vasodilatation in atherosclerotic arteries.

Iron-depleting nutritional immunity controls extracellular bacterial replication in Legionella pneumophila infections

The accidental human pathogen Legionella pneumophila (Lp) is the etiological agent for a severe atypical pneumonia known as Legionnaires’ disease. In human infections and animal models of disease alveolar macrophages are the primary cellular niche that supports bacterial replication within a unique intracellular membrane-bound organelle. The Dot/Icm apparatus—a type IV secretion system that translocates ~300 bacterial proteins within the cytosol of the infected cell—is a central virulence factor required for intracellular growth. Mutant strains lacking functional Dot/Icm apparatus are transported to and degraded within the lysosomes of infected macrophages. The early foundational work from Dr. Horwitz’s group unequivocally established that Legionella does not replicate extracellularly during infection—a phenomenon well supported by experimental evidence for four decades. Our data challenges this paradigm by demonstrating that macrophages and monocytes provide the necessary nutrients and support robust Legionella extracellular replication. We show that the previously reported lack of Lp extracellular replication is not a bacteria intrinsic feature but rather a result of robust restriction by serum-derived nutritional immunity factors. Specifically, the host iron-sequestering protein Transferrin is identified here as a critical suppressor of Lp extracellular replication in an iron-dependent manner. In iron-overload conditions or in the absence of Transferrin, Lp bypasses growth restriction by IFNγ-primed macrophages though extracellular replication. It is well established that certain risk factors associated with development of Legionnaires’ disease, such as smoking, produce a chronic pulmonary environment of iron-overload. Our work indicates that iron-overload could be an important determinant of severe infection by allowing Lp to overcome nutritional immunity and replicate extracellularly, which in turn would circumvent intracellular cell intrinsic host defenses. Thus, we provide evidence for nutritional immunity as a key underappreciated host defense mechanism in Legionella pathogenesis.

Single–particle rotational microrheology enables pathological staging of macrophage foaming and antiatherosclerotic studies

The formation of macrophage-derived foam cells has been recognized as the pathological hallmark of atherosclerotic diseases. However, the pathological evolution dynamics and underlying regulatory mechanisms remain largely unknown. Herein, we introduce a single-particle rotational microrheology method for pathological staging of macrophage foaming and antiatherosclerotic explorations by probing the dynamic changes of lysosomal viscous feature over the pathological evolution progression. The principle of this method involves continuous monitoring of out-of-plane rotation-caused scattering brightness fluctuations of the gold nanorod (AuNR) probe-based microrheometer and subsequent determination of rotational relaxation time to analyze the viscous feature in macrophage lysosomes. With this method, we demonstrated the lysosomal viscous feature as a robust pathological reporter and uncovered three distinct pathological stages underlying the evolution dynamics, which are highly correlated with a pathological stage-dependent activation of the NLRP3 inflammasome-involved positive feedback loop. We also validated the potential of this positive feedback loop as a promising therapeutic target and revealed the time window-dependent efficacy of NLRP3 inflammasome-targeted drugs against atherosclerotic diseases. To our knowledge, the pathological staging of macrophage foaming and the pathological stage-dependent activation of the NLRP3 inflammasome-involved positive feedback mechanism have not yet been reported. These findings provide insights into in-depth understanding of evolutionary features and regulatory mechanisms of macrophage foaming, which can benefit the analysis of effective therapeutical drugs as well as the time window of drug treatment against atherosclerotic diseases in preclinical studies.

Modulation of anti-cardiac fibrosis immune responses by changing M2 macrophages into M1 macrophages

BackgroundMacrophages play a crucial role in the development of cardiac fibrosis (CF). Although our previous studies have shown that glycogen metabolism plays an important role in macrophage inflammatory phenotype, the role and mechanism of modifying macrophage phenotype by regulating glycogen metabolism and thereby improving CF have not been reported.MethodsHere, we took glycogen synthetase kinase 3β (GSK3β) as the target and used its inhibitor NaW to enhance macrophage glycogen metabolism, transform M2 phenotype into anti-fibrotic M1 phenotype, inhibit fibroblast activation into myofibroblasts, and ultimately achieve the purpose of CF treatment.ResultsNaW increases the pH of macrophage lysosome through transmembrane protein 175 (TMEM175) and caused the release of Ca2+ through the lysosomal Ca2+ channel mucolipin-2 (Mcoln2). At the same time, the released Ca2+ activates TFEB, which promotes glucose uptake by M2 and further enhances glycogen metabolism. NaW transforms the M2 phenotype into the anti-fibrotic M1 phenotype, inhibits fibroblasts from activating myofibroblasts, and ultimately achieves the purpose of treating CF.ConclusionOur data indicate the possibility of modifying macrophage phenotype by regulating macrophage glycogen metabolism, suggesting a potential macrophage-based immunotherapy against CF.Graphical

Transcriptional and ultrastructural changes of macrophages after african swine fever virus infection

African swine fever (ASF) is a highly impactful infectious disease in the swine industry, leading to substantial economic losses globally. The causative agent, African swine fever virus (ASFV), possesses intricate pathogenesis, warranting further exploration. In this study, we investigated the impact of ASFV infection on host gene transcription and organelle changes through macrophage transcriptome sequencing and ultrastructural transmission electron microscopy observation. According to the results of the transcriptome sequencing, ASFV infection led to significant alterations in the gene expression pattern of porcine bone marrow derived macrophages (BMDMs), with 2404 genes showing upregulation and 1579 genes downregulation. Cytokines, and chemokines were significant changes in the expression of BMDMs; there was significant activation of pattern recognition receptors such as Toll-like receptors and Nod-like receptors. According to the observation of the ultrastructure, mitochondrial damage and mitochondrial autophagy were widely present in ASFV-infected cells. The reduced number of macrophage pseudopodia suggested that virus-induced structural changes may compromise pathogen recognition, phagocytosis, and signal communication in macrophages. Additionally, the decreased size and inhibited acidification of secondary lysosomes in macrophages implied suppressed phagocytosis. Overall, ASFV infection resulted in significant changes in the expression of cytokines and chemokines, accompanied by the activation of NLR and TLR signaling pathways. We reported for the first time that ASFV infection led to a reduction in pseudopodia numbers and a decrease in the size and acidification of secondary lysosomes.

Gallic acid promotes macrophage phagosome acidification and phagolysosome formation by activating NLRP3/mTOR signaling pathway

IntroductionGallic acid (GA) has a good therapeutic effect in bacteriological inhibition and plays a variety of functions in maintaining the stability of the immune system. The aim of the present study was to investigate the effect of GA on the bactericidal activity of macrophages against Vibrio vulnificus (Vv). MethodsA cell counting kit-8 (CCK-8) assay was carried out to test the cytotoxicity of GA on J774A.1 cells. Concentration of proinflammatory cytokines in J774A.1 cells were evaluated by ELISA. The internalization and degradation of Vv in the phagosomes were observed by transmission electron microscopy (TEM). The phagosome acidification and phagolysosome formation were detected to evaluate the bacteria-clearing function of J774A.1 cells. The bactericidal activity of GA in vivo was also investigated by collecting the survival time of Vv infected mice and observing the inflammatory infiltration of organs. ResultsOur results demonstrated that GA at 50 μM significantly inhibited the proinflammatory cytokines levels, promoted phagosome acidification and phagolysosome formation in J774A.1 cells with Vv infection. This may be related to the activation of NLRP3/mTOR signaling pathway. Additionally, GA treatment improves the survival and bactericidal activity of mice infected with Vv. ConclusionsIn summary, GA exerts bactericidal activity against Vv infection by regulating the formation and acidification of phagocytic lysosomes in macrophages.

BODIPY-Based NIR Trackers with Acidity-Driven Amphiphilicity for STED Super-Resolution Imaging of Lysosomal Membranes.

Although super-resolution imaging provides a great opportunity to disclose the structures of living cells at the nanoscale level, resolving the structural details of organelles is highly dependent on the targeting accuracy and photophysical properties of fluorescence trackers. Herein, we report a series of ultrabright and photostable trackers of lysosomal membranes for super-resolution imaging using stimulated emission depletion microscopy (STED). These trackers are composed of lipophilic NIR BODIPY derivatives and ionizable tertiary amines. This structural feature enables accurate targeting of the lysosomal membrane through the formation of transient amphiphilicity driven by the acidity in the lysosome. As a representative, Lyso-700 is applied for STED-based super-resolution imaging of the lysosomal membrane of living macrophages. By use of Lyso-700, the interaction details between lysosomes of macrophages and fungi are visualized. Overall, these trackers display great potential as advanced lysosome trackers and merit further evaluation for lysosome-related studies.

Imipramine Treatment Alters Sphingomyelin, Cholesterol, and Glycerophospholipid Metabolism in Isolated Macrophage Lysosomes.

Lysosomes are degradative organelles that facilitate the removal and recycling of potentially cytotoxic materials and mediate a variety of other cellular processes, such as nutrient sensing, intracellular signaling, and lipid metabolism. Due to these central roles, lysosome dysfunction can lead to deleterious outcomes, including the accumulation of cytotoxic material, inflammation, and cell death. We previously reported that cationic amphiphilic drugs, such as imipramine, alter pH and lipid metabolism within macrophage lysosomes. Therefore, the ability for imipramine to induce changes to the lipid content of isolated macrophage lysosomes was investigated, focusing on sphingomyelin, cholesterol, and glycerophospholipid metabolism as these lipid classes have important roles in inflammation and disease. The lysosomes were isolated from control and imipramine-treated macrophages using density gradient ultracentrifugation, and mass spectrometry was used to measure the changes in their lipid composition. An unsupervised hierarchical cluster analysis revealed a clear differentiation between the imipramine-treated and control lysosomes. There was a significant overall increase in the abundance of specific lipids mostly composed of cholesterol esters, sphingomyelins, and phosphatidylcholines, while lysophosphatidylcholines and ceramides were overall decreased. These results support the conclusion that imipramine's ability to change the lysosomal pH inhibits multiple pH-sensitive enzymes in macrophage lysosomes.

OP38 Evaluation Of A High-Cost Medicine For A Rare Disease: 16-Year Cohort Of Imiglucerase Use For Gaucher Disease In Brazil

IntroductionGaucher disease is a lysosomal storage disease of autosomal recessive inheritance that is caused by a deficiency of the enzyme glucocerebrosidase. This deficiency results in accumulation of the enzyme’s main substrate in the lysosomes of macrophages, mainly in the spleen, liver, and bone marrow. In more severe cases it can affect the lung, kidneys, and central nervous system. There are two main treatments available for patients with Gaucher disease: enzyme replacement therapy and inhibition of substrate synthesis. The main enzyme replacement therapy used in Brazil is imiglucerase, an analog of the human β-glucocerebrosidase enzyme. Imiglucerase is produced by recombinant DNA technology using a cell culture derived from the Chinese hamster ovary. It has 497 amino acids and differs from the endogenous enzyme by an amino acid at position 495, where histidine is replaced by arginine. The objective of the study was to analyze the survival of patients treated for Gaucher disease with imiglucerase in Brazil from 2000 to 2015.MethodsWe constructed a retrospective cohort study of patients with Gaucher disease who received imiglucerase through the Brazilian National Health System from 2000 to 2015 using a national database created from the linkage of administrative databases.ResultsA total of 1,241 patients who received imiglucerase were included. The overall survival rates at one, ten, and 15 years were 98.7 percent (95% confidence interval [CI]: 98.1, 99.4), 92.3 percent (95% CI: 90.2, 94.4), and 89.4 percent (95% CI: 85.6, 93.3), respectively.ConclusionsOur findings advance the understanding of the profile, survival, and risk factors of people with Gaucher disease, adding new data to the discussion regarding pharmaceutical therapies and patient care, and providing data for the development of new public health policies for the use of advanced, high-cost drugs for rare diseases.

Pulmonary tuberculosis biomarker miR-215-5p inhibits autophagosome-lysosome fusion in macrophages

The normal autophagy flux is beneficial for the rapid elimination of phagocytic pathogens by macrophages. However, Mycobacterium tuberculosis interferes with the autophagy flux of macrophages to weaken their immune function and evade immune surveillance. In this study, we found that miRNA-215-5p was downregulated in tuberculosis patients. A potential diagnostic model for tuberculosis was established by combining miRNA-215-5p with three others differentially expressed microRNAs (miRNA-145-5p, miRNA-486-5p and miRNA-628-3p). Furthermore, we discovered that the up-regulated miRNA-215-5p could inhibit the maturation of autophagy by preventing the fusion of autophagosomes with lysosomes in macrophages. The role of TB-specific miRNA-215-5p in inhibiting auto-lysosome formation provides evidence of its potential role in Host-directed therapy for tuberculosis.

Oxidized cholesteryl ester induces exocytosis of dysfunctional lysosomes in lipidotic macrophages.

A key event in atherogenesis is the formation of lipid-loaded macrophages, lipidotic cells, which exhibit irreversible accumulation of undigested modified low-density lipoproteins (LDL) in lysosomes. This event culminates in the loss of cell homeostasis, inflammation, and cell death. Nevertheless, the exact chemical etiology of atherogenesis and the molecular and cellular mechanisms responsible for the impairment of lysosome function in plaque macrophages are still unknown. Here, we demonstrate that macrophages exposed to cholesteryl hemiazelate (ChA), one of the most prevalent products of LDL-derived cholesteryl ester oxidation, exhibit enlarged peripheral dysfunctional lysosomes full of undigested ChA and neutral lipids. Both lysosome area and accumulation of neutral lipids are partially irreversible. Interestingly, the dysfunctional peripheral lysosomes are more prone to fuse with the plasma membrane, secreting their undigested luminal content into the extracellular milieu with potential consequences for the pathology. We further demonstrate that this phenotype is mechanistically linked to the nuclear translocation of the MiT/TFE family of transcription factors. The induction of lysosome biogenesis by ChA appears to partially protect macrophages from lipid-induced cytotoxicity. In sum, our data show that ChA is involved in the etiology of lysosome dysfunction and promotes the exocytosis of these organelles. This latter event is a new mechanism that may be important in the pathogenesis of atherosclerosis.

Argon gas plasma-treated physiological solutions stimulate immunogenic cell death and eradicates immunosuppressive CD47 protein in lung carcinoma.

Cold atmospheric plasma-treated liquids (PTLs) exhibit selective toxicity toward tumor cells and are provoked by a cocktail of reactive oxygen and nitrogen species in such liquids. Compared to the gaseous phase, these reactive species are more persistent in the aqueous phase. This indirect plasma treatment method has gradually gathered interest in the discipline of plasma medicine to treat cancer. PTL's motivated effect on immunosuppressive proteins and immunogenic cell death (ICD) in solid cancer cells is still not explored. In this study, we aimed to induce immunomodulation by plasma-treated Ringer's lactate (PT-RL) and phosphate-buffered saline (PT-PBS) solutions for cancer treatment. PTLs induced minimum cytotoxicity in normal lung cells and inhibited cancer cell growth. ICD is confirmed by the enhanced expression of damage-associated molecular patterns (DAMPs). We evidenced that PTLs induce intracellular nitrogen oxide species accumulation and elevate immunogenicity in cancer cells owing to the production of pro-inflammatory cytokines, DAMPs, and reduced immunosuppressive protein CD47 expression. In addition, PTLs influenced A549cells to elevate the organelles (mitochondria and lysosomes) in macrophages. Taken together, we have developed a therapeutic approach to potentially facilitate the selection of a suitable candidate for direct clinical applications.

Particulate air pollution exaggerates diet-induced insulin resistance through NLRP3 inflammasome in mice

Air particulate matter 2.5 (PM2.5) has been demonstrated to exaggerate insulin resistance in both human and animal studies. However, the exact molecular mechanisms remain elusive. This study sought to assess the role of NLRP3 inflammasome in PM2.5 exposure-induced insulin resistance and explore the underlying mechanisms. Wild-type (WT), Nlrp3−/−, Tlr4Lps-d, or Nrf2−/− mice, on a normal diet or high-fat diet (HFD), were exposed to PM2.5 or filtered air (FA) in a whole-body exposure facility. Priming (first signal) and assembly (second signal) of NLRP3 inflammasome activation were assessed by measuring the transcription of Nlrp3/Il-1β and detecting the activity of caspase-1 and secretion of IL-1β. We found PM2.5 exposure exaggerated insulin resistance and increased IL-1β production in the HFD-fed WT mice, but not Nlrp3−/− mice. Gene expressions of Nlrp3 and Il-1β in the lungs and peritoneal macrophages were upregulated in WT mice exposed to PM2.5. When stimulated with LPS (first signal) or monosodium urate (second signal), PM2.5 exposure was able to enhance the activity of caspase-1 and IL-1β secretion, suggesting that PM2.5 may serve as a stimulus of either the first or second signal for NLRP3 inflammasome activation. Effects of PM2.5 on caspase-1 activation and IL-1β secretion were partially blocked in Tlr4Lps-d mice. Reactive oxygen species (ROS), co-localization of NLRP3 and mitochondria, and secondary lysosomes in macrophages were increased after PM2.5 exposure, while deficiency of antioxidant gene Nrf2 in mice significantly enhanced PM2.5-induced secretion of IL-1β. Imaging flow cytometry and transmission electron microscopy demonstrated an engulfment of PM2.5 particles by macrophages, while suppression of phagocytosis by cytochalasin D abolished PM2.5-induced transcription of Nlrp3/Il-1β. Our results demonstrated a critical role of NLRP3 inflammasome in PM2.5 exaggerated insulin resistance, and multiple pathways in the first and second signals of NLRP3 inflammasome activation may be involved.

The Effects of Ginkgo biloba Extract on Autophagy in Human Macrophages Stimulated by Cigarette Smoke Extract.

To investigate the effects of Ginkgo biloba extract (GBE) on autophagy in human macrophages stimulated by cigarette smoke extract (CSE). The human monocyte cell line U937 was cultured in vitro, and phorbol ester (PMA) was added to the cell culture medium to induce differentiation into human macrophages. CSE was prepared by traditional methods for experiments. The cells were divided into four groups: the blank group, the CSE model group, the GBE + CSE group, and the rapamycin + CSE group. Immunofluorescence was used to identify human macrophages, transmission electron microscopy was used to observe the ultrastructure of human macrophages in each group, ELISA was used to measure the amount of IL-6 and IL-10 in the supernatant from each group of cells, the mRNA levels of p62, ATG5, ATG7, and Rab7 were measured by real-time qPCR, and the protein expression levels of p62, ATG5, ATG7, and Rab7 were measured by Western blotting. U937 cells were successfully differentiated into human macrophages after induction with PMA. The CSE model group had many more autophagosomes than the blank group. Compared with the CSE model group, the GBE + CSE group and the rapamycin + CSE group had significantly more autophagolysosomal. Compared with the other groups, the CSE model group had a higher level of IL-6 but a lower level of IL-10 in the supernatant (p < 0.05). Compared with the blank group, the mRNA and protein expression levels of p62 in the CSE model group were significantly decreased, while the mRNA and protein expression levels of ATG5 and ATG7 were significantly increased in the CSE model group (p < 0.05). No difference was found in the mRNA and protein expression levels of Rab7 between the blank group and the CSE model group. Compared with the CSE model group, the IL-6 level in the GBE + CSE group and the rapamycin + CSE group cell culture supernatant decreased significantly, p62 mRNA and protein expression significantly decreased, while ATG5, ATG7, and Rab7 mRNA and protein expression levels were significantly increased (p < 0.05). Moreover, increased LC3-II/LC3-I ratio were also found in the GBE + CSE group and the rapamycin + CSE group compared with the CSE model group. GBE could promote the fusion of autophagosomes and lysosomes in human macrophages, enhance the autophagy function of human macrophages, and reduce the damaging effect of CSE on the autophagy function of macrophages.

A 20-Year Longitudinal Study of Plasma Chitotriosidase Activity in Treated Gaucher Disease Type 1 and 3 Patients-A Qualitative and Quantitative Approach.

Chitotriosidase is an enzyme produced and secreted in large amounts by activated macrophages, especially macrophages loaded with phagocytozed glycosphingolipid in Gaucher disease. Macrophages phagocytose decayed blood cells that contain a lot of sphingolipid-rich cell membranes. In Gaucher disease, due to a deficit in beta-glucocerebrosidase activity, the phagocytozed substrate glucocerebroside cannot undergo further catabolism. In such a situation, macrophages secrete chitotriosidase in proportion to the degree of overload. Gaucher disease (GD) is a recessively inherited disorder resulting in storage of glucosylceramide (GlcCer) in lysosomes of tissue macrophages. It is directly caused by the deficiency of beta-glucocerebrosidase (GBA) activity. Chitotriosidase has been measured systematically each year in the same group of 49 patients with type 1 and 3 GD for over 20 years. Our analysis showed that chitotriosidase is very sensitive biomarker to enzyme replacement therapy (ERT). The response to treatment introduction is of an almost immediate nature, lowering pathologically high chitotriosidase levels by a factor of 2 in a time scale of 8 months, on average. Long term enzyme replacement therapy (ERT) brings chitotriosidase activity close to reference values. Finally, reducing the dose of ERT quickly boosts chitotriosidase activity, but restoring the initial dose of treatment brings chitotriosidase level of activity back onto the decreasing time trajectory.

Gaucher disease protects against tuberculosis

Biallelic mutations in the glucocerebrosidase (GBA1) gene cause Gaucher disease, characterized by lysosomal accumulation of glucosylceramide and glucosylsphingosine in macrophages. Gaucher and other lysosomal diseases occur with high frequency in Ashkenazi Jews. It has been proposed that the underlying mutations confer a selective advantage, in particular conferring protection against tuberculosis. Here, using a zebrafish Gaucher disease model, we find that the mutation GBA1 N370S, predominant among Ashkenazi Jews, increases resistance to tuberculosis through the microbicidal activity of glucosylsphingosine in macrophage lysosomes. Consistent with lysosomal accumulation occurring only in homozygotes, heterozygotes remain susceptible to tuberculosis. Thus, our findings reveal a mechanistic basis for protection against tuberculosis by GBA1 N370S and provide biological plausibility for its selection if the relatively mild deleterious effects in homozygotes were offset by significant protection against tuberculosis, a rampant killer of the young in Europe through the Middle Ages into the 19th century.

Specific preadaptations of Rhodococcus equi cooperate with its Virulence-associated protein A during macrophage infection.

Gram-positive Rhodococcus equi (Prescotella equi) is a lung pathogen of foals and immunocompromised humans. Intra-macrophage multiplication requires production of the bacterial Virulence-associated protein A (VapA) which is released into the phagosome lumen. VapA pH-neutralizes intracellular compartments allowing R. equi to multiply in an atypical macrophage phagolysosome. Here, we show that VapA does not support intra-macrophage growth of several other bacterial species demonstrating that only few bacteria have the specific preadaptations needed to profit from VapA. We show that the closest relative of R. equi, environmental Rhodococcus defluvii (Prescotella defluvii), does not multiply in macrophages at 37°C even when VapA is present because of its thermosensitivity but it does so once the infection temperature is lowered providing rare experimental evidence for 'thermal restriction'. Using growth experiments with isolated macrophage lysosomes and modified infection schemes we provide evidence that R. equi resists the attack by phagolysosome contents at low pH for several hours. During this time, R. equi produces and secretes VapA which enables it to grow at the expense of lysosome constituents. We present arguments that, under natural infection conditions, R. equi is VapA-less during the initial encounter with the host. This has important implications for vaccine development.

Use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in atherosclerosis

ABSTRACT Dysfunction in the macrophage lysosomal system including reduced acidity and diminished degradative capacity is a hallmark of atherosclerosis, leading to blunted clearance of excess cellular debris and lipids in plaques and contributing to lesion progression. Devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles have emerged as an effective platform to both target specific tissues and serve as drug delivery vehicles. In most cases, administered nanoparticles are taken up non-selectively by the mononuclear phagocyte system including monocytes/macrophages leading to the undesirable degradation of cargo in lysosomes. We took advantage of this default route to target macrophage lysosomes to rectify their acidity in disease states such as atherosclerosis. Herein, we develop and test two commonly used acidic nanoparticles, poly-lactide-co-glycolic acid (PLGA) and polylactic acid (PLA), both in vitro and in vivo. Our results in cultured macrophages indicate that the PLGA-based nanoparticles are the most effective at trafficking to and enhancing acidification of lysosomes. PLGA nanoparticles also provide functional benefits including enhanced lysosomal degradation, promotion of macroautophagy/autophagy and protein aggregate removal, and reduced apoptosis and inflammasome activation. We demonstrate the utility of this system in vivo, showing nanoparticle accumulation in, and lysosomal acidification of, macrophages in atherosclerotic plaques. Long-term administration of PLGA nanoparticles results in significant reductions in surrogates of plaque complexity with reduced apoptosis, necrotic core formation, and cytotoxic protein aggregates and increased fibrous cap formation. Taken together, our data support the use of acidic nanoparticles to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis. Abbreviations: BCA: brachiocephalic arteries; FACS: fluorescence activated cell sorting; FITC: fluorescein-5-isothiocyanatel; IL1B: interleukin 1 beta; LAMP: lysosomal associated membrane protein; LIPA/LAL: lipase A, lysosomal acid type; LSDs: lysosomal storage disorders; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence intensity; MPS: mononuclear phagocyte system; PEGHDE: polyethylene glycol hexadecyl ether; PLA: polylactic acid; PLGA: poly-lactide-co-glycolic acid; SQSTM1/p62: sequestosome 1

Effects of metformin and simvastatin treatment on ultrastructural features of liver macrophages in HFD mice

ABSTRACT Type 2 diabetes is a major health burden to the society. Macrophages and liver inflammation emerged as important factors in its development. We investigated ultrastructural changes in the liver, with a special emphasis on macrophages in high fat diet (HFD) fed C57BL/6 J mice treated with metformin or simvastatin, two drugs that are used frequently in diabetes. Both metformin and simvastatin reduced the liver damage in HFD fed animals, manifested as the prevention of nonalcoholic steatohepatitis development and reduced activation and number of macrophages in the liver, as well as the percentage of these cells with lipid droplets in the cytoplasm compared to untreated HFD animals. In contrast with untreated HFD-fed animals, lipid droplets were not observed in lysosomes of macrophages in HFD animals treated with metformin and simvastatin. These findings provide new insight into the effects of metformin and simvastatin on the liver in this experimental model of type 2 diabetes and provide further rationale for implementation of statins in the therapeutic regimens in this disease.

Pitfalls in methods to study colocalization of nanoparticles in mouse macrophage lysosomes

BackgroundIn the field of nanoscience there is an increasing interest to follow dynamics of nanoparticles (NP) in cells with an emphasis on endo-lysosomal pathways and long-term NP fate. During our research on this topic, we encountered several pitfalls, which can bias the experimental outcome. We address some of these pitfalls and suggest possible solutions. The accuracy of fluorescence microscopy methods has an important role in obtaining insights into NP interactions with lysosomes at the single cell level including quantification of NP uptake in a specific cell type.MethodsHere we use J774A.1 cells as a model for professional phagocytes. We expose them to fluorescently-labelled amorphous silica NP with different sizes and quantify the colocalization of fluorescently-labelled NP with lysosomes over time. We focus on confocal laser scanning microscopy (CLSM) to obtain 3D spatial information and follow live cell imaging to study NP colocalization with lysosomes.ResultsWe evaluate different experimental parameters that can bias the colocalization coefficients (i.e., Pearson’s and Manders’), such as the interference of phenol red in the cell culture medium with the fluorescence intensity and image post-processing (effect of spatial resolution, optical slice thickness, pixel saturation and bit depth). Additionally, we determine the correlation coefficients for NP entering the lysosomes under four different experimental set-ups. First, we found out that not only Pearson’s, but also Manders’ correlation coefficient should be considered in lysosome-NP colocalization studies; second, there is a difference in NP colocalization when using NP of different sizes and fluorescence dyes and last, the correlation coefficients might change depending on live-cell and fixed-cell imaging set-up.ConclusionsThe results summarize detailed steps and recommendations for the experimental design, staining, sample preparation and imaging to improve the reproducibility of colocalization studies between the NP and lysosomes.Graphical