Macrophage Reaction Research Articles

DDR1 activation in macrophage promotes IPF by regulating NLRP3 inflammasome and macrophage reaction

BackgroundDiscoidin Domain Receptor1 (DDR1) is a member of receptor tyrosine kinases (RTKs) which have been reported to be associated with idiopathic pulmonary fibrosis (IPF), but the mechanism remains unclear. MethodsBleomycin-induced IPF mice model was performed in this study, and two DDR1 inhibitors were administered in vivo, to investigate the role of DDR1 in IPF. Lentivirus mediated DDR1-/- stable Raw264.7 macrophage cell line or DDR1 inhibitors treatment in vitro, to study the effect of DDR1 on inflammasome activation and macrophage responses. All of the mechanisms were further tested in the lung sections of IPF patients. ResultHere, we reported that: (i) Both specific inhibitors of DDR1 dramatically alleviated the symptoms of bleomycin-induced IPF models. (ii) Immunofluorescence staining showed that DDR1 signaling is activated in macrophages. In vivo molecular biological analysis proved that DDR1 activation exacerbates IPF inflammation through inflammasome signaling, macrophage activation, and M1/M2 polarization. (iii) Extracellular matrix (ECM) such as Collagen 1 activates DDR1 in macrophage cell line Raw264.7 in vitro, to mediate inflammasome activation and macrophage responses. (iv) DDR1 activation in macrophage was confirmed in IPF patients’ samples, which could be one of the mechanisms for the pathogenesis of IPF. DiscussionIn this study, we firstly reported DDR1 activation in macrophages to play a role in IPF via inflammasome activation and macrophage responses. In addition, DDR1 inhibitors DDR1-IN-1 and DDR1-IN-2 exerted significant anti-inflammatory and anti-fibrotic effects in IPF, all of which provide a potentially effective therapeutic medication for clinical IPF treatment.

Electroconductive PEDOT nanoparticle integrated scaffolds for spinal cord tissue repair

BackgroundHostile environment around the lesion site following spinal cord injury (SCI) prevents the re-establishment of neuronal tracks, thus significantly limiting the regenerative capability. Electroconductive scaffolds are emerging as a promising option for SCI repair, though currently available conductive polymers such as polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) present poor biofunctionality and biocompatibility, thus limiting their effective use in SCI tissue engineering (TE) treatment strategies.MethodsPEDOT NPs were synthesized via chemical oxidation polymerization in miniemulsion. The conductive PEDOT NPs were incorporated with gelatin and hyaluronic acid (HA) to create gel:HA:PEDOT-NPs scaffolds. Morphological analysis of both PEDOT NPs and scaffolds was conducted via SEM. Further characterisation included dielectric constant and permittivity variances mapped against morphological changes after crosslinking, Young’s modulus, FTIR, DLS, swelling studies, rheology, in-vitro, and in-vivo biocompatibility studies were also conducted.ResultsIncorporation of PEDOT NPs increased the conductivity of scaffolds to 8.3 × 10–4 ± 8.1 × 10–5 S/cm. The compressive modulus of the scaffold was tailored to match the native spinal cord at 1.2 ± 0.2 MPa, along with controlled porosity. Rheological studies of the hydrogel showed excellent 3D shear-thinning printing capabilities and shape fidelity post-printing. In-vitro studies showed the scaffolds are cytocompatible and an in-vivo assessment in a rat SCI lesion model shows glial fibrillary acidic protein (GFAP) upregulation not directly in contact with the lesion/implantation site, with diminished astrocyte reactivity. Decreased levels of macrophage and microglia reactivity at the implant site is also observed. This positively influences the re-establishment of signals and initiation of healing mechanisms. Observation of axon migration towards the scaffold can be attributed to immunomodulatory properties of HA in the scaffold caused by a controlled inflammatory response. HA limits astrocyte activation through its CD44 receptors and therefore limits scar formation. This allows for a superior axonal migration and growth towards the targeted implantation site through the provision of a stimulating microenvironment for regeneration.ConclusionsBased on these results, the incorporation of PEDOT NPs into Gel:HA biomaterial scaffolds enhances not only the conductive capabilities of the material, but also the provision of a healing environment around lesions in SCI. Hence, gel:HA:PEDOT-NPs scaffolds are a promising TE option for stimulating regeneration for SCI.

Effects of transplanted mesenchymal stem cells on repair of the lung tissue of rats with experimental pulmonary fibrosis

Pulmonary fibrosis is one of the commonest forms of interstitial lung diseases with poorly studied methods of its treatment in both human and veterinary medicines. Therefore, this paper focused on seeking alternative methods of its diagnostics and treatment. The article provides the results of the study of bronchoalveolar lavage fluid of rats with experimental lung fibrosis and influence of transplanted allogeneic mesenchymal stem cells of the bone marrow on stimulation of regenerative processes in damaged lung tissues. The studies were conducted on female Wistar rats with pulmonary fibrosis modeled using single transthoracic injection of solution of bleomycin hydrochloride. For the purpose of treatment, we used allogeneic mesenchymal stem cells introduced by various methods and the traditional treatment. We determined that best normalization of the parameters of the studied brochoalveolar lavage occurred in animals that received mesenchymal stem cells. The most active repair processes were in the experimental group that received the mesenchymal stem cells directly to the lung tissue. The animals that received intravenous injection of mesenchymal stemm cells were observed to have lower clinical parameters of the brochoalveolar lavage, but still better than such in the group treated traditionally. The lowest parameters were in animals that received the traditional treatment; they were greater than the phisological parameters, but significantly exceeded them in animals of the control group, indicating presence of inflammatory process in the lung tissue. The conducted cytological assays of the samples of the brochoalveolar lavage revealed that experimental animals with experimental pulmonary fibrosis had development of macrophage and lymphocytic reactions under the influence of transplanted mesenchymal stemm cells. We observed no atypical cells in all the experimental groups. This allows us to draw a conclusion that using stem cells by various methods of transplantation does not stimulate the onset of negative reactons (formation of atypical cells, metastatic processes, etc). Thus, the results of the study of the influence of transplanted mesenchymal stem cells demonstrate that in the conditions of experimental pulmonary fibrosis, the activity of regenerative processes in pathologically altered lung tissue may be an effective method of treatment of animals with this kind of pathology.

In vitro two-step granuloma formation model for testing innate immune response to implants and coatings

The extensive innate immune response to implanted biomaterials contributes significantly to their sub-par performance and failure. Granuloma formation is one of such reactions which results in multi-cell type clusters in line with the immune reaction to implanted materials. However, currently no in vitro model of granuloma formation exists that takes into account the arrival of multiple cell types (immune cells and connective tissue cells) to the implant insertion site. In this study, we developed a two-step model based on stimulated macrophage seeding followed by fibroblast introduction after a physiologically relevant time period for mimicking initial steps of immune reaction to biomaterials and inducing granuloma like behavior. Both LPS and TNF-α induction resulted in granuloma like formations which persisted longer than the control conditions. Introduction of human fibroblasts resulted in the colonization of the surfaces where the cell numbers and the collagen secretion were dependent on the microenvironment. In order to demonstrate the capacity of our model system to monitor the reaction to a given coating, a validated antimicrobial coating (Polyarginine (PAR)/Hyaluronic acid (HA)) was used as a testing bed. The coating prevented the adhesion of macrophages while allowing the adhesion of the fibroblast at the time of their arrival. Similar to its antimicrobial activity, macrophage metabolic activity and M2 differentiation in the presence of PAR was dependent to its chain length. The incorporation of fibroblasts resulted in decreased TNF-α and increased IL-1RA secretion especially in stimulation conditions. The pro- and anti-inflammatory cytokine secretions were low for PAR/HA coatings in line with the decreased number of macrophage presence. In the presence of complex PBMC population, the coating resulted in slightly less cellular attachment, without any significant cytokine secretion; the absence of inflammatory reaction was also demonstrated in vivo in a mouse model. The described in vitro granuloma testing system can control the macrophage reaction as a function of stimulation. It can also be used for testing new biomaterials for the potential innate immune responses and also for validation of implant coatings beyond their primary function from the immune response point of view.

A Postmortem Analysis of Polyethylene Damage and Periprosthetic Tissue in Rotating Platform and Fixed Bearing Tibial Inserts.

Mobile bearing designs are intended to reduce wear, but mixed results were reported from retrieval analyses. Postmortem evaluation (PM) provides the opportunity to assess polyethylene damage in successful implants. We compared damage patterns, MRI presentation, and histology between mobile-bearing and fixed tibial inserts retrieved postmortem and compared these results to our prior findings from implants retrieved at revision. Eleven postmortem knees with rotating platform (RP) implants and 13 with fixed bearing (FB) implants were examined. All were MRI scanned, and tissue samples were collected from standardized regions for histology. Polyethylene inserts were subjectively scored to assess articular, backside, and PS post surfaces for damage modes and severity. Average duration of implantation was 9.3 years (1.7-19.6 years). Surface burnishing was the most common polyethylene damage mode. Average damage scores were higher for RP (53.4) compared to FB inserts (34.4) due to greater backside damage (13.4 for RP vs 1.4 for FB). A minimal difference in damage was observed on the articular surfaces (37.4 RP vs 30.0 FB). Mild innate macrophage reactions were seen in 8 (72.7%) RP and 5 (45.5%) FB specimens. Polyethylene particles were identified in 7 (63.6%) RP and 3 (27.7%) FB specimens. Postmortem inserts showed low damage levels and mild tissue reactions compared to those reported for implants removed at revision arthroplasty. Nonetheless, trends in comparing RP and FB inserts were consistent with those seen in retrieval analyses, demonstrating the usefulness of retrieval studies in capturing performance differences among TKA designs.

Tea Ingredients Have Anti-coronavirus Disease 2019 (COVID-19) Targets Based on Bioinformatics Analyses and Pharmacological Effects on LPS-Stimulated Macrophages.

Coronavirus disease 2019 (COVID-19) is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused millions of deaths and lacks treatment. Although several studies have focused on the major component of green tea, epigallocatechin 3-gallate (EGCG), which is efficient in preventing COVID-19, systemic analyses of the anti-COVID-19 potential of green tea remain insufficient. Here, we co-analyzed the target genes of tea ingredients and COVID-19 signature genes and found that epigallocatechin 3-acetalbehyde was capable of reversing the major molecular processes of COVID-19 (MAPK and NF-κB activation). These findings were further supported by Western blotting (WB), immunofluorescence, and quantitative polymerase chain reaction (qPCR) in LPS-stimulated macrophages. Moreover, using molecular docking analysis, we identified three tea ingredients ((-)-catechin gallate, D-(+)-cellobiose, and EGCG) that may interact with the vital SARS-CoV-2 protein, 5R84, compared with the qualified 5R84 ligand WGS. Thus, our results indicated that tea ingredients have the potential to treat COVID-19 by suppressing the COVID-19 signature genes and interacting with the vital SARS-CoV-2 protein.

Effect of exercise from a very early stage after intracerebral hemorrhage on microglial and macrophage reactivity states in rats.

This study investigated the effects of exercise, starting very early after intracerebral hemorrhage (ICH), on microglia and macrophages in a rat model. Collagenase solution was injected into the left striatum to induce ICH. Rats were randomly assigned to receive placebo surgery without exercise (sham surgery), ICH without exercise (ICH), or ICH with very early exercise (ICH + VET). The ICH + VET group was subjected to treadmill running 6 h, 24 h, and days 2-6 after ICH. Motor function assessment was performed using the ladder test and rotarod test 3 h, 25 h, and 7 days after ICH. Postexercise brain tissue was collected on day 8 after surgery to investigate the lesion volume. Very early exercise temporarily worsened motor dysfunction. The protein expression levels of the macrophage and microglial markers CD80, CD163, and TMEM119 were analyzed 6 h, 24 h, and 8 days after ICH. Protein analysis of NeuN, GFAP, and PSD95 was also performed on day 8 after ICH. There was no significant difference in lesion volume between the ICH and ICH + VET groups on day 8 after ICH. Exercise from very early stage prevented elevated CD163 protein expression. Very early exercise may inhibit the activation of anti-inflammatory-associated macrophages/microglia.

Effect of naturally derived surgical hemostatic materials on the proliferation of A549 human lung adenocarcinoma cells.

Hemostatic materials are generally applied in surgical operations for cancer, but their effects on the growth and recurrence of tumors are unclear. Herein, three commonly used naturally derived hemostatic materials, gelatin sponge, Surgicel (oxidized regenerated cellulose), and biopaper (mixture of sodium hyaluronate and carboxymethyl chitosan), were cocultured with A549 human lung adenocarcinoma cells in vitro. Furthermore, the performance of hemostatic materials and the tumorigenicity of the materials with A549 ​cells were observed after subcutaneous implantation into BALB/c mice. The in vitro results showed that biopaper was dissolved quickly, with the highest cell numbers at 2 and 4 days of culture. Gelatin sponges retained their structure and elicited the least cell infiltration during the 2- to 10-day culture. Surgicel partially dissolved and supported cell growth over time. The in vivo results showed that biopaper degraded rapidly and elicited an acute Th1 lymphocyte reaction at 3 days after implantation, which was decreased at 7 days after implantation. The gelatin sponge resisted degradation and evoked a hybrid M1/M2 macrophage reaction at 7–21 days after implantation, and a protumor M2d subset was confirmed. Surgicel resisted early degradation and caused obvious antitumor M2a macrophage reactions. Mice subjected to subcutaneous implantation of A549 ​cells and hemostatic materials in the gelatin sponge group had the largest tumor volumes and the shortest overall survival (OS), while the Surgicel and the biopaper group had the smallest volumes and the longest OS. Therefore, although gelatin sponges exhibited cytotoxicity to A549 ​cells in vitro, they promoted the growth of A549 ​cells in vivo, which was related to chronic M2d macrophage reaction. Surgicel and biopaper inhibited A549 ​cell growth in vivo, which is associated with chronic M2a macrophage reaction or acute Th1 lymphocyte reaction.

Identification of the possible negative impact of the drilling reagent brucit on workers involved in the construction of wells

In the technological processes of the construction of oil and gas wells, a variety of general-purpose inorganic chemicals are used. The ingress of these reagents into the body of drilling workers together with inhaled air, food intake, through the pores and mucous membrane is currently not sufficiently studied. The work investigated the effect of the drilling reagent brucit on the lung tissue of rats. During inhalation administration of brucite to rats, dystrophic processes were noted in their lungs, which were accompanied by the development of chronic inflammation, acute macrophage reaction. Morphometric analysis also showed a significant decrease in the area of the alveolar space and the area of the bronchus of medium caliber. In the lungs, the formation of many airless areas alternating with emphysematous-altered, the severity of which increased with the duration of exposure to the drilling reagent. Scientific novelty: In the work, the early unexplored effect of the drilling reagent brucit on the lung tissue of rats was determined. The categories of workers exposed to brucite at all stages of production are determined.For the first time, morphological features of the lungs in rats were determined against the background of inhaled administration of brucite particles. Based on the results of the study, the toxicological characteristics of the drilling reagent brucit are given. Research results:Brucite particles present in the air, when inhaled, passed through the nose, trachea, bronchi and reached the alveoli of non-linearalbinorats rats. Obviously, part of the administered dose was excreted by the body with excrement and mucus, and the other part was absorbed and then distributed to organs and tissues. A control section of a medium-caliber bronchus is shown in Figure 2. 14 days after the introduction of brucite particles, changes in lung tissue can be seen on microphotographs of the histological section. Focal fullness of the capillaries of the interalveolar septa is revealed. The changes registered on the 14th day after the introduction of brucite particles are shown in Figure 3.

The effect of different sizes of cross‐linked fibers of biodegradable electrospun poly(ε‐caprolactone) scaffolds on osteogenic behavior in a rat model in vivo

AbstractA variety of biodegradable polymer scaffolds serving the purpose of mimicking the extracellular matrix have been studied; however, no effective recipe has yet been developed for scaffold pore size and overall percent porosity, which are optimal for cell penetration in vitro and tissue growth in vivo. Thus, this study reports the effect of poly(ε‐caprolactone) (PCL) fiber size and scaffold porosity on the osteogenic behavior of scaffolds in a rat model in vivo. It was revealed that scaffolds with an average fiber size of 9.2 μm, and 4.05 μm, and combination of 0.6 μm submicrofibres (upper layer) with 9.2 μm microfibres (bottom layer) in hybrid two‐layer scaffolds behave significantly differently with respect to macrophage reactions and tissue regeneration in a rat model in vivo. Poor macrophage response at implantation tests into the bone tissue in vivo reveals the advantages of microfibrous PCL scaffolds with a fiber size of 4.05 μm and hybrid scaffolds with submicro‐ and microfibres for stimulation of reparative osteogenesis over microfibrous scaffolds with a fiber size of 9.2 μm. The combination of submicro‐ and microfibrous layers in hybrid scaffolds provides an opportunity to control the shape and density of bone trabeculae during regeneration processes in vivo.

Abstract TP14: Translational Modeling Of Morphological And Physiological Features Of Vascular Dementia: A Review

Vascular dementia (VaD) is the second-most common form of dementia, accounting for 20% of all dementia cases. Although the pathobiology of VaD is poorly understood and lacks effective treatment, clinically relevant factors such as age and sex are rarely considered in preclinical modeling. This places efforts to develop future therapies at a severe disadvantage, as clinically relevant translation of underlying pathobiology may be lost. We reviewed common morphological and physiological outcomes in six major rodent models of VaD in a total of 258 full-text publications: 1) chronic cerebral hypoperfusion, 2) high fat diet, 3) diabetes, 4) hypertension, 5) carotid arterial calcification, and 6) and CADASIL (Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy), and evaluated translational features relevant to human pathophysiology with an analysis of timepoints of observed pathology and rodent sex and age. We found that all models of VaD shared common features of decreased CBF and upregulation of inflammatory signaling molecules such as TNF-α, IL-1β, and IL-6, and reactive oxygen species (ROS) such as SOD and NOX, consistent with clinical presentations of VaD. Only the carotid artery calcification model showed inconsistent evidence for brain endothelial tight junction loss, astrogliosis, and macrophage reactivity, in disagreement with other models. We conclude that improved translational insight may critically depend on 1) inclusion of a constellation of comorbidities in preclinical modeling instead of modeling each in isolation to better capture human disease pathogenesis, 2) increased use of middle-aged and aged preclinical rodent models which are sensitive to clinically relevant disease presentation in middle-age and elderly individuals, and 3) inclusion of female animals in disease models as sex is a relevant biological factor which may better define future therapeutic strategy in real-world populations.

Ferric Ammonium Citrate Upregulates PD-L1 Expression through Generation of Reactive Oxygen Species.

Iron plays an important role in macrophage polarization by altering metabolic and redox status. However, the impact of iron on the immune status of macrophages is still controversial. In this study, we report that ferric ammonium citrate (FAC) upregulates PD-L1 expression in macrophages. FAC not only altered the phenotype of macrophages but also led to enriching immune-modulatory T cell subsets. Since iron is known to be a constituent of coenzymes facilitating metabolic processes in mitochondria, we examined the metabolic status of FAC-overloaded macrophages by measuring the oxygen consumption rate (OCR) and the represented coenzyme, aconitase. In addition to enhancement of metabolic processes, FAC accelerated the Fenton reaction in macrophages, which also contributed to the facilitation of oxygen consumption. We reasoned that the enhancement of the OCR leads to the production of reactive oxygen species (ROS), which are directly linked to PD-L1 induction. Using ferrostatin, rotenone, and N-acetyl-L-cysteine, we confirmed that metabolic and redox regulation is responsible for FAC-mediated PD-L1 expression. Furthermore, we suggested that FAC-induced ROS production may explain FAC-mediated pro- and anti-inflammatory responses in macrophages. These findings may extend our understanding of regulating iron concentration during immune checkpoint therapy in cancer patients.

Prognostic Role of Tumor Microenvironment in DLBCL and Relation to Patients' Clinical Outcome: A Clinical and Immunohistochemical Study.

Diffuse large B cell lymphoma is the most common type of lymphoma in Egypt with an unfavorable prognosis. The tumor microenvironment is rich in immune response either T cells or macrophages. The current study is aimed at testing CD4, CD8, CD68, and MMP9 immunohistochemistry of DLBCL activities with the prognosis of the tumor. The results showed no positive relation between T cell and macrophage reaction to the tumor prognosis suggesting that this reaction is part of the tumor process and not a defense mechanism from the surrounding stroma.

Modulation of the inflammatory response to decellularized collagen matrix for cartilage regeneration.

Decellularized extracellular matrices (DECM) are among the most common types of materials used in tissue engineering due to their cell instructive properties, biodegradability, and accessibility. Particularly in cartilage, a natural collagen type II matrix can be a promising means to provide the necessary cues and support for chondrogenic stem and progenitor cells (CSPCs). However, efficient remodeling of the transplanted DECM is largely dependent on the host immune response, with macrophages playing the central role in orchestrating both inflammatory and regenerative processes. Here we assessed the reaction of human primary macrophages to the cartilage DECM. Our findings show that the xenogeneic collagen matrix can elicit a mixed response in human macrophages, whereby the inflammatory response (M1) and the activation of remodeling (M2) type of macrophages are both present. Additionally, we demonstrate the inhibitory effect of macrophage response on the migratory capacity of human CSPCs. We further show that the inflammatory reaction of macrophages to the cartilage DECM, as well as the resulting inhibitory effects on CSPC migration, can be attenuated by interleukin-4 (IL-4). Finally, we demonstrate that IL-4 can effectively bind the matrix, thereby modulating macrophage response by reducing the inflammatory reaction and inducing the M2 phenotype.

Biometry in Silicone Oil Filled Eyes. A Review

The “gold standard” of modern vitreoretinal surgery is silicone oil tamponade of the vitreous cavity. The lens opacity development is in the list of complications of prolonged silicone oil eye filling (from 2 weeks to 2 years). Polydimethylsiloxanes hydrophobicity, direct contact with the front of the silicone bladder, macrophage and toxic reaction, trophic disturbances are the causes leading to the cataract initiation. This makes the problem of cataract surgery and preliminary intraocular lens calculation in silicone oil filled eyes before its removing very relevant as well as cloudy retina visualization and the necessity of minimization of number of operations through their combination. Certainly, the main error in IOL power calculation is associated with axial length measurement inaccuracy, as the most significant term of an equation. Silicone oil filled eyes biometry errors, and, consequently, postoperative refraction biases remain unresolved problem until now. To date authors report only 58 % of cases in which target refraction was achieved after combined surgery. Some researchers figure out that average calculation error after phacoemulsification with IOL implantation in avitreal eyes was 0.8 D despite of the optical biometry usage. Today it is represented by several methods: partial coherent interferometry, optical low-coherence reflectometry and optical coherence tomography, which are implemented in devices such as IOLMaster 500, Lenstar LS 900 and IOLMaster 700, which have their own characteristics and measurement accuracy. Their advantages as well as creation an accurate IOL calculation method for silicone oil filled eyes could reduce postoperative refraction error that outline significant medical and social problem.

Pinocembrin ameliorates acute liver failure via activating the Sirt1/PPARα pathway in vitro and in vivo

Acute liver failure (ALF) is a life-threatening disease and affects multiple organ systems. Pro-inflammatory factors derived from macrophage plays a key role in septicemia. Pinocembrin is a natural favonoid compound, which can be extracted from honey, propolis and several other plants. Recent investigations demonstrate that Pinocembrin has a variety of pharmacological activities, including anti-inflammatory and antioxidant. To investigate the effects of Pinocembrin on ALF, we explored its possible molecular mechanisms through the experiments in vivo and in vitro. Pre-treatment with Pinocembrin attenuated LPS-induced hepatocyte dysfunction and reduced levels of pro-inflammatory factors and macrophages infiltration. Pinocembrin inhibited the hepatocyte apoptosis and pro-inflammatory reaction of peritoneal macrophages by reducing reactive oxygen species (ROS) via the Sirt1/PPARα signaling pathway. Our study suggests that Pinocembrin might represent a novel therapeutic drug and offers a new method for the treatment of ALF.

Exosomes derived from inflammatory myoblasts promote M1 polarization and break the balance of myoblast proliferation/differentiation.

BACKGROUNDAcute muscle injuries are one of the most common injuries in sports. Severely injured muscles are prone to re-injury due to fibrotic scar formation caused by prolonged inflammation. How to regulate inflammation and suppress fibrosis is the focus of promoting muscle healing. Recent studies have found that myoblasts and macrophages play important roles in the inflammatory phase following muscle injury; however, the crosstalk between these two types of cells in the inflammatory environment, particularly the exosome-related mechanisms, had not been well studied. AIMTo evaluate the effects of exosomes from inflammatory C2C12 myoblasts (IF-C2C12-Exos) on macrophage polarization and myoblast proliferation/differentiation.METHODSA model of inflammation was established in vitro by lipopolysaccharide stimulation of myoblasts. C2C12-Exos were isolated and purified from the supernatant of myoblasts by gradient centrifugation. Multiple methods were used to identify the exosomes. Gradient concentrations of IF-C2C12-Exos were added to normal macrophages and myoblasts. PKH67 fluorescence tracing was used to identify the interaction between exosomes and cells. Microscopic morphology, Giemsa stain, and immunofluorescence were carried out for histological analysis. Additionally, ELISA assays, flow cytometry, and western blot were conducted to analyze molecular changes. Moreover, myogenic proliferation was assessed by the BrdU test, scratch assay, and CCK-8 assay.RESULTSWe found that the PKH-67-marked C2C12-Exos can be endocytosed by both macrophages and myoblasts. IF-C2C12-Exos induced M1 macrophage polarization and suppressed the M2 phenotype in vitro. In addition, these exosomes also stimulated the inflammatory reactions of macrophages. Furthermore, we demonstrated that IF-C2C12-Exos disrupted the balance of myoblast proliferation/differentiation, leading to enhanced proliferation and suppressed fibrogenic/myogenic differentiation.CONCLUSIONIF-C2C12-Exos can induce M1 polarization, resulting in a sustained and aggravated inflammatory environment that impairs myoblast differentiation, and leads to enhanced myogenic proliferation. These results demonstrate why prolonged inflammation occurs after acute muscle injury and provide a new target for the regulation of muscle regeneration.

Morphofunctional changes in the tissue of the brain, liver and kidneys of white rats under the influence of selenium nanocomposite encapsulated in the polymer matrix of arabinogalactan

Introduction. Due to their high biocompatibility, substances based on nanosized selenium particles, encapsulated in natural or synthetic polymer matrices, are promising materials for the creation of biomedical preparations of diagnostic and therapeutic value. Selenium nanoparticles are successfully used in the diagnosis of various types of cancer. In addition to the diagnostic value, selenium nanoparticles have their own prophylactic and oncological effect. This paper presents the results of a study of the toxicity of the Se nanocomposite encapsulated in the polymer matrix of arabinogalactan (SeAG). The emergence and development of the pathological process in the tissue of the brain, liver and kidneys during subacute administration of this nanocomposite was studied.Materials and methods. Twenty white outbred male rats weighing 200–220 g were used in the work. Animals were orally administered a solution of the selenium nanocomposite at a dose of 500 μg per kilogram of animal body weight for 10 days. Then, using the methods of histological analysis, the severity of the biological response of the organism to the introduction of this nanocomposite was assessed. An analysis of the state of the tissue of the liver, kidneys and the sensorimotor cortex of the brain was carried out.Results. With the intragastric administration of this drug, there is stasis of blood in the portal tracts, a pronounced macrophage reaction and diapedesis of leukocytes in the liver tissue. There is a decrease in the number of normal neurons per unit area, a decrease in the number of astroglia cells and an increase in the number of degeneratively altered neurons in the tissue of the sensorimotor cortex. There is also an increase in connective tissue in the cortex of the kidney, with the formation of fibrosis and a decrease in the area of the Shumlyansky – Bowman capsule.Conclusion. The effect of the investigated nanocomposite is characterized by the development of a pronounced pathological process in the central nervous and hepatorenal systems of the body.

Innate Memory Reprogramming by Gold Nanoparticles Depends on the Microbial Agents That Induce Memory.

Innate immune memory, the ability of innate cells to react in a more protective way to secondary challenges, is induced by exposure to infectious and other exogeous and endogenous agents. Engineered nanoparticles are particulate exogenous agents that, as such, could trigger an inflammatory reaction in monocytes and macrophages and could therefore be also able to induce innate memory. Here, we have evaluated the capacity of engineered gold nanoparticles (AuNPs) to induce a memory response or to modulate the memory responses induced by microbial agents. Microbial agents used were in soluble vs. particulate form (MDP and the gram-positive bacteria Staphylococcus aureus; β-glucan and the β-glucan-producing fungi C. albicans), and as whole microrganisms that were either killed (S. aureus, C. albicans) or viable (the gram-negative bacteria Helicobacter pylori). The memory response was assessed in vitro, by exposing human primary monocytes from 2-7 individual donors to microbial agents with or without AuNPs (primary response), then resting them for 6 days to allow return to baseline, and eventually challenging them with LPS (secondary memory response). Primary and memory responses were tested as production of the innate/inflammatory cytokine TNFα and other inflammatory and anti-inflammatory factors. While inactive on the response induced by soluble microbial stimuli (muramyl dipeptide -MDP-, β-glucan), AuNPs partially reduced the primary response induced by whole microorganisms. AuNPs were also unable to directly induce a memory response but could modulate stimulus-induced memory in a circumscribed fashion, limited to some agents and some cytokines. Thus, the MDP-induced tolerance in terms of TNFα production was further exacerbated by co-priming with AuNPs, resulting in a less inflammatory memory response. Conversely, the H. pylori-induced tolerance was downregulated by AuNPs only relative to the anti-inflammatory cytokine IL-10, which would lead to an overall more inflammatory memory response. These effects of AuNPs may depend on a differential interaction/association between the reactive particle surfaces and the microbial components and agents, which may lead to a change in the exposure profiles. As a general observation, however, the donor-to-donor variability in memory response profiles and reactivity to AuNPs was substantial, suggesting that innate memory depends on the individual history of exposures.

Functional role of galectin-9 in directing human innate immune reactions to Gram-negative bacteria and T cell apoptosis

Galectin-9 is a member of the galectin family of proteins, which were first identified to specifically bind to carbohydrates containing β-galactosides. Galectin-9 is conserved through evolution and recent evidence demonstrated its involvement in innate immune reactions to bacterial infections as well as the suppression of cytotoxic immune responses of T and natural killer cells. However, the molecular mechanisms underlying such differential immunological functions of galectin-9 remain largely unknown. In this work we confirmed that soluble galectin-9 derived from macrophages binds to Gram-negative bacteria by interacting with lipopolysaccharide (LPS), which forms their cell wall. This opsonisation effect most likely interferes with the mobility of bacteria leading to their phagocytosis by innate immune cells. Galectin-9-dependent opsonisation also promotes the innate immune reactions of macrophages to these bacteria and significantly enhances the production of pro-inflammatory cytokines – interleukin (IL) 6, IL-1β and tumour necrosis factor alpha (TNF-α). In contrast, galectin-9 did not bind peptidoglycan (PGN), which forms the cell wall of Gram-positive bacteria. Moreover, galectin-9 associated with cellular surfaces (studied in primary human embryonic cells) was not involved in the interaction with bacteria or bacterial colonisation. However, galectin-9 expressed on the surface of primary human embryonic cells, as well as soluble forms of galectin-9, were able to target T lymphocytes and caused apoptosis in T cells expressing granzyme B. Furthermore, “opsonisation” of T cells by galectin-9 led to the translocation of phosphatidylserine onto the cell surface and subsequent phagocytosis by macrophages through Tim-3, the receptor, which recognises both galectin-9 and phosphatidylserine as ligands.