Phagocytosis Activity Research Articles

Purified granulocytes in extracorporeal cell therapy: A multifaceted approach to combat sepsis-induced immunoparalysis.

Immune cell dysfunction plays a central role in sepsis-induced immunoparalysis. Targeted treatment using healthy donor immune cell transfusions, particularly granulocyte concentrates (GC) potentially induces tissue damage. Initial trials using GC in an extracorporeal immune cell perfusion system provided evidence for beneficial effects with fewer side effects, by separating patient and donor immune cell compartments. A multicenter clinical trial is exploring feasibility and effects of a 6-h treatment (NCT06143137). This ex vivo study examines technical feasibility and cellular effects of an extended treatment interval up to 24 h. Standard GC were purified to increase the potential storage time and subsequently implemented in the extracorporeal immune cell perfusion system. Parameters assessed included cell viability, phagocytosis activity, oxidative burst, cytokine release, and metabolic parameters of purified. GC during an extended circulation time of up to 24 h. After storage of 72 h granulocytes were viable throughout the study period and exhibited preserved functionality and metabolic activity. The findings highlight a time-dependent nature of cytokine release by neutrophils in the extracorporeal circuit, as cytokine secretion patterns showed IL-8 peaking within 6 h, while MCP-1, IL-6, IL-1β, and TNF-α increased after 24 h of circulation. Purified GC remain functional after 72 h of storage and additional 24 h in the circulating treatment model. Cytokine secretion patterns revealed a significant increase, especially between 10 and 24 h of treatment. Extending treatment time holds promise for enhancing immune response against sepsis-induced immunoparalysis. These findings provide valuable insights for optimizing immune-targeted therapeutic interventions.

Tumor necrosis factor regulates leukocyte recruitment but not bacterial persistence during Staphylococcus aureus craniotomy infection

BackgroundCraniotomy is a common neurosurgery used to treat intracranial pathologies. Nearly 5% of the 14 million craniotomies performed worldwide each year become infected, most often with Staphylococcus aureus (S. aureus), which forms a biofilm on the surface of the resected bone segment to establish a chronic infection that is recalcitrant to antibiotics and immune-mediated clearance. Tumor necrosis factor (TNF), a prototypical proinflammatory cytokine, has been implicated in generating protective immunity to various infections. Although TNF is elevated during S. aureus craniotomy infection, its functional importance in regulating disease pathogenesis has not been explored.MethodsA mouse model of S. aureus craniotomy infection was used to investigate the functional importance of TNF signaling using TNF, TNFR1, and TNFR2 knockout (KO) mice by quantifying bacterial burden, immune infiltrates, inflammatory mediators, and transcriptional changes by RNA-seq. Complementary experiments examined neutrophil extracellular trap formation, leukocyte apoptosis, phagocytosis, and bactericidal activity.ResultsTNF transiently regulated neutrophil and granulocytic myeloid-derived suppressor cell recruitment to the brain, subcutaneous galea, and bone flap as evident by significant reductions in both cell types between days 7 to 14 post-infection coinciding with significant decreases in several chemokines, which recovered to wild type levels by day 28. Despite these defects, bacterial burdens were similar in TNF KO and WT mice. RNA-seq revealed enhanced lymphotoxin-α (Lta) expression in TNF KO granulocytes. Since both TNF and LTα signal through TNFR1 and TNFR2, KO mice for each receptor were examined to assess potential redundancy; however, neither strain had any impact on S. aureus burden. In vitro studies revealed that TNF loss selectively altered macrophage responses to S. aureus since TNF KO macrophages displayed significant reductions in phagocytosis, apoptosis, IL-6 production, and bactericidal activity in response to live S. aureus, whereas granulocytes were not affected.ConclusionThese findings implicate TNF in modulating granulocyte recruitment during acute craniotomy infection via secondary effects on chemokine production and identify macrophages as a key cellular target of TNF action. However, the lack of changes in bacterial burden in TNF KO animals suggests the involvement of additional signals that dictate S. aureus pathogenesis during craniotomy infection.

Macrophages allocate before apoptosis initiation and produce reactive oxygen species during interdigital phagocytosis.

During programmed cell death (PCD), it is commonly accepted that macrophages are recruited by apoptotic cells to complete cell degradation. Interdigital cell death, a classical model of PCD, contributes to digit individualization in limbs of mammals and other vertebrates. Here, we show that macrophages are present in interdigits before significant cell death occurs and remain after apoptosis inhibition. The typical interdigital phagocytic activity was not observed after a partial depletion of macrophages and was markedly reduced by engulfment/phagosome maturation inhibition, as detected by its association with high lysosomal activity. β-galactosidase activity in this region was also coupled with phagocytosis, against its relationship with cellular senescence. Interdigital phagocytosis correlated with high levels of reactive oxygen species (ROS), common in embryo regions carrying abundant cell death, suggesting that macrophages are the major source of ROS. ROS generation was dependent on NADPH oxidases and blood vessel integrity, but not directly associated with lysosomal activity. Therefore, macrophages prepattern regions where abundant cell death is going to occur, and high lysosomal activity and the generation of ROS by an oxidative burst-like phenomenon are activities of phagocytosis.

Oligodeoxynucleotides containing CpG motifs upregulate bactericidal activities of heterophils and enhance immunoprotection of neonatal broiler chickens against Salmonella Typhimurium septicemia

In the past, we demonstrated that oligodeoxynucleotides containing CpG motifs (CpG-ODN) mimicking bacterial DNA, stimulate the innate immune system of neonatal broiler chickens and protect them against Escherichia coli and Salmonella Typhimurium (S. Typhimurium) septicemia. The first line of innate immune defense mechanism is formed by heterophils and plays a critical protective role against bacterial septicemia in avian species. Therefore, the objectives of this study were 1) to explore the kinetics of CpG-ODN mediated antibacterial mechanisms of heterophils following single or twice administration of CpG-ODN in neonatal broiler chickens and 2) to investigate the kinetics of the immunoprotective efficacy of single versus twice administration of CpG-ODN against S. Typhimurium septicemia. In this study, we successfully developed and optimized flow cytometry-based assays to measure phagocytosis, oxidative burst, and degranulation activity of heterophils. Birds that received CpG-ODN had significantly increased (p < 0.05) phagocytosis, oxidative burst, and degranulation activity of heterophils as early as 24 h following CpG-ODN administration. Twice administration of CpG-ODN significantly increased the phagocytosis activity of heterophils. In addition, our newly developed CD107a based flow cytometry assay demonstrated a significantly higher degranulation activity of heterophils following twice than single administration of CpG-ODN. However, the oxidative burst activity of heterophils was not significantly different between birds that received CpG-ODN only once or twice. Furthermore, delivery of CpG-ODN twice increased immunoprotection against S. Typhimurium septicemia compared to once but the difference was not statistically significant. In conclusion, we demonstrated enhanced bactericidal activity of heterophils after administration of CpG-ODN to neonatal broiler chickens. Further investigations will be required to identify other activated innate immune cells and the specific molecular pathways associated with the CpG-ODN mediated activation of heterophils.

Novel insights into paclitaxel’s role on tumor-associated macrophages in enhancing PD-1 blockade in breast cancer treatment

BackgroundTriple-negative breast cancer (TNBC) poses unique challenges due to its complex nature and the need for more effective treatments. Recent studies showed encouraging outcomes from combining paclitaxel (PTX) with programmed...

Bioengineer mesenchymal stem cell for treatment of glioma by IL‐12 mediated microenvironment reprogramming and nCD47‐SLAMF7 mediated phagocytosis regulation of macrophages

AbstractHigh expression of cellular self‐activated immunosuppressive molecules and extensive infiltration of suppressive immune cells in the tumor microenvironment are the main factors contributing to glioma's resistance to immunotherapy. Nonetheless, technology to modify the expression of glioma cellular self‐molecules through gene editing requires further development. This project advances cell therapy strategies to reverse the immunosuppressive microenvironment of glioma (TIME). Bone marrow‐derived mesenchymal stem cells (MSCs) are engineered to express bioactive proteins and demonstrate tumor‐homing characteristics upon activation by TGF‐β. These MSCs are designed to secrete the anti‐tumor immune cytokine IL‐12 and the nCD47‐SLAMF7 fusion protein, which regulates T‐cell activity and macrophage phagocytosis. The engineered MSCs are then injected in situ into the glioma site, circumventing the blood‐brain barrier to deliver high local concentrations of bioactive proteins. This approach aims to enhance the M1 polarization of infiltrating macrophages, stimulate macrophage‐mediated tumor cell phagocytosis, activate antigen‐presenting cells, and promote effector CD8+ T cell infiltration, effectively controlling glioma. Additionally, the engineered MSCs may serve as a universal treatment for other tumors that express TGF‐β at high levels. This study proposes a novel treatment strategy for the clinical management of glioma patients.

Effect of Giving Api-Api (Avicennia marina ) Mangrove Leaf Solution In Artificial Feed On The Immunity Response and Growth of Vannamei Shrimp (Litopenaeus vannamei)

The purpose of this study was .to determine the effect of giving Avicennia marina mangrove leaf solution on artificial feed to increase the immune response and growth of vannamei shrimp. The parameters observed in this study were the enhancement of PA value (Phagocytosis Activity), ADG (Average Daily Growth), FCR (Feed Conversion Ratio), and EPP (Efficiency of Feed Utilization). The vannamei shrimp used was PL-20 fry with the weight of 1,09 – 1,13 gâ‹…head-1 and density 1 shrimp L-1. The method used in this study was an experimental method in the laboratory, with a completely randomized design (CRD) with 4 treatments and 3 replications. This research was conducted by adding Avicennia marina mangrove leaf solution to the feed, with treatments of A (control), B (125 "°), C (175 "°), and D (225 "°). The addition of Avicennia marina leaf solution to the feed had a significant effect on the increase of phagocytosis activity and growth of the shrimp with F count of 30,773 which was higher than the F table of 4,06. Moreover, the best dose obtained in treatment D which resulted in an increase in PA was 61,9%, ADG was 0,22 gâ‹…day-1, FCR was 1,29, and EPP was 79,04%.

Selective depletion of tumor-infiltrating regulatory T cells with BAY 3375968, a novel Fc-optimized anti-CCR8 antibody

Regulatory T cells (Tregs) are known to facilitate tumor progression by suppressing CD8+ T cells within the tumor microenvironment (TME), thereby also hampering the effectiveness of immune checkpoint inhibitors (ICIs). While systemic depletion of Tregs can enhance antitumor immunity, it also triggers undesirable autoimmune responses. Therefore, there is a need for therapeutic agents that selectively target Tregs within the TME without affecting systemic Tregs. In this study, as shown also by others, the chemokine (C–C motif) receptor 8 (CCR8) was found to be predominantly expressed on Tregs within the TME of both humans and mice, representing a unique target for selective depletion of tumor-residing Tregs. Based on this, we developed BAY 3375968, a novel anti-human CCR8 antibody, along with respective surrogate anti-mouse CCR8 antibodies, and demonstrated their in vitro mode-of-action through induction of potent antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) activities. In vivo, anti-mouse CCR8 antibodies effectively depleted Tregs within the TME primarily via ADCP, leading to increased CD8+ T cell infiltration and subsequent tumor growth inhibition across various cancer models. This monotherapeutic efficacy was significantly enhanced in combination with ICIs. Collectively, these findings suggest that CCR8 targeting represents a promising strategy for Treg depletion in cancer therapies. BAY 3375968 is currently under investigation in a Phase I clinical trial (NCT05537740).

Adaptive immune response in women from the Russian Arctic region after COVID-19 infection

BACKGROUND: The Arctic region of Russia is characterized by disproportionately high rates of morbidity and mortality from COVID-19 during the pandemic. The harsh climatic and environmental conditions in this area impede the development of self-regulation processes resulting in activation and strain of both cellular and humoral immunity. This leads to a depletion of the body’s reserve capacities. At present, there is lack of research examining how individuals who have recovered from COVID-19 are affected by the extreme conditions of Arctic Russia. AIM: To study the ratio of immunocompetent cells involved in the adaptive immune response following COVID-19 infection. MATERIAL AND METHODS: A total of 29 women aged 20 – 40 years were examined in Arkhangelsk as part of a comprehensive immunological study. This study involved assessment of the number of leukocytes, lymphocytes, and their phenotypes (CD5+, CD8+, CD10+, CD95+), as well as determination of phagocytic activity and phagocytic number. RESULTS: The cellular adaptive immune response in observed individuals 6 months after experiencing moderate COVID-19 disease was characterized by a very low concentration of T cells (CD5+) in all cases, CD10+ lymphocytes (44.83%) alongside with a high concentration of cytotoxic lymphocytes (CD8+) in 48.27% of individuals and lymphocytes with receptors for apoptosis (CD95+) in 51.72%, with relatively high phagocytic activity ranging from 90 to 100%. A correlation was found in 11.29% of women between the low content of CD10+ and CD95+ cells with the activity of phagocytosis. In 40% of women with high phagocytic activity, the concentrations of cytotoxic cells (CD8+) were found to be at a minimum level. CONCLUSIONS: Women with high phagocytic activity were found to have the lowest concentrations of cytotoxic cells, suggesting a potentially positive prognosis for reducing the risk of complications. This indicates that cellular immunity may play a role in determining the severity of COVID-19 infection in individuals with high phagocytic activity.

Dimorphic Regulation of the MafB Gene by Sex Steroids in Hamsters, Mesocricetus auratus.

MafB is a transcription factor that regulates macrophage differentiation. Macrophages are a traditional feature of the hamster Harderian gland (HG); however, studies pertaining to MafB expression in the HG are scant. Here, the full-length cDNA of the MafB gene in hamsters was cloned and sequenced. Molecular characterization revealed that MafB encodes a protein containing 323 amino acids with a DNA-binding domain, a transactivation domain, and a leucine zipper domain. qPCR assays indicated that MafB was expressed in different tissues of both sexes. The highest relative expression levels in endocrine tissues were identified in the pancreas. Gonadectomy in male hamsters was associated with significantly higher mRNA levels in the HG; replacement with dihydrotestosterone restored mRNA expression. The HG in male hamsters contained twofold more MafB mRNA than the HG of female hamsters. Adrenals revealed similar mRNA relative expression levels during the estrous cycle. The estrous phase was associated with higher mRNA levels in the ovary. A significantly up-regulated expression and sexual dimorphism of MafB was found in the pancreas. Therefore, MafB in the HG may play an active role in the macrophage differentiation required for phagocytosis activity and intraocular repair. Additionally, sex steroids appear to strongly influence the MafB expression in the HG and pancreas. These studies highlight the probable biological importance of MafB in immunological defense and pancreatic β cell regulation.

Immunomodulatory Effects of Ethanol Extract of Lime Peel on Phagocytosis Activity and Delayed-Type Hypersensitivity Response

The natural human environment is inhabited by various organisms, including those harmful to the body such as bacteria, viruses, fungi, and other pathogens. This condition weakens the immune system's response to pathogens, making the body more susceptible to diseases such as rheumatoid arthritis, cancer, and other inflammations. In some cases, this condition can be fatal, leading to death. This study aims to examine the immunomodulatory effects of Ethanol Extract of Lime Peel on phagocytosis activity and delayed-type hypersensitivity response in experimental animals. The research methods include sample collection and processing, plant identification, extract preparation, and immunomodulatory effect testing. Phytochemical screening results showed the presence of alkaloids, glycosides, flavonoids, tannins, and steroids in the extract of the lime peel. Simplicia and ethanol extract of lime peel characterization yielded results meet the standards of the Indonesian Herbal Pharmacopoeia 2nd edition. Immunomodulatory effect testing was conducted through phagocytosis activity and delayed-type hypersensitivity response assays. The results showed that the extract at doses of 25, 50, and 100 mg/kgBW had immunostimulatory effects in enhancing phagocytosis activity, while doses of 17.5 mg/kgBW, 35 mg/kgBW, and 70 mg/kgBW significantly increased delayed-type hypersensitivity response compared to the negative control. These findings indicate the potential of ethanol extract of Citrus aurantifolia as an immunomodulator that can enhance the body's immune system. This study provides an important contribution to the development of immunomodulatory therapy derived from natural ingredients such as lime peel for managing immunity-related diseases with lower side effects.

A Perspective on the CD47-SIRPA Axis in High-Risk Neuroblastoma.

Neuroblastoma is a pediatric cancer with significant clinical heterogeneity. Despite extensive efforts, it is still difficult to cure children with high-risk neuroblastoma. Immunotherapy is a promising approach to treat children with this devastating disease. We have previously reported that macrophages are important effector cells in high-risk neuroblastoma. In this perspective article, we discuss the potential function of the macrophage inhibitory receptor SIRPA in the homeostasis of tumor-associated macrophages in high-risk neuroblastoma. The ligand of SIRPA is CD47, known as a "don't eat me" signal, which is highly expressed on cancer cells compared to normal cells. CD47 is expressed on both tumor and stroma cells, whereas SIRPA expression is restricted to macrophages in high-risk neuroblastoma tissues. Notably, high SIRPA expression is associated with better disease outcome. According to the current paradigm, the interaction between CD47 on tumor cells and SIRPA on macrophages leads to the inhibition of tumor phagocytosis. However, data from recent clinical trials have called into question the use of anti-CD47 antibodies for the treatment of adult and pediatric cancers. The restricted expression of SIRPA on macrophages in many tissues argues for targeting SIRPA on macrophages rather than CD47 in CD47/SIRPA blockade therapy. Based on the data available to date, we propose that disruption of the CD47-SIRPA interaction by anti-CD47 antibody would shift the macrophage polarization status from M1 to M2, which is inferred from the 1998 study by Timms et al. In contrast, the anti-SIRPA F(ab')2 lacking Fc binds to SIRPA on the macrophage, mimics the CD47-SIRPA interaction, and thus maintains M1 polarization. Anti-SIRPA F(ab')2 also prevents the binding of CD47 to SIRPA, thereby blocking the "don't eat me" signal. The addition of tumor-opsonizing and macrophage-activating antibodies is expected to enhance active tumor phagocytosis.

Immunomodulation by glucocorticoid-induced leucine zipper in macrophages: enhanced phagocytosis, protection from pyroptosis, and altered mitochondrial function.

Glucocorticoids, which have long served as fundamental therapeutics for diverse inflammatory conditions, are still widely used, despite associated side effects limiting their long-term use. Among their key mediators is glucocorticoid-induced leucine zipper (GILZ), recognized for its anti-inflammatory and immunosuppressive properties. Here, we explore the immunomodulatory effects of GILZ in macrophages through transcriptomic analysis and functional assays. Bulk RNA sequencing of GILZ knockout and GILZ-overexpressing macrophages revealed significant alterations in gene expression profiles, particularly impacting pathways associated with the inflammatory response, phagocytosis, cell death, mitochondrial function, and extracellular structure organization activity. GILZ-overexpression enhances phagocytic and antibacterial activity against Salmonella typhimurium and Escherichia coli, potentially mediated by increased nitric oxide production. In addition, GILZ protects macrophages from pyroptotic cell death, as indicated by a reduced production of reactive oxygen species (ROS) in GILZ transgenic macrophages. In contrast, GILZ KO macrophages produced more ROS, suggesting a regulatory role of GILZ in ROS-dependent pathways. Additionally, GILZ overexpression leads to decreased mitochondrial respiration and heightened matrix metalloproteinase activity, suggesting its involvement in tissue remodeling processes. These findings underscore the multifaceted role of GILZ in modulating macrophage functions and its potential as a therapeutic target for inflammatory disorders, offering insights into the development of novel therapeutic strategies aimed at optimizing the benefits of glucocorticoid therapy while minimizing adverse effects.

Study of Macrophage Activity in Cats with FIP and Naturally FCoV-Shedding Healthy Cats.

Coronavirus frequently infects humans and animals, showing the ability to recombine and cross over to different species. Cats can be considered a model for studying coronavirus infection, in which feline coronavirus (FCoV) represents a major enteric pathogen related to gastroenteric disease. In this animal, the virus can acquire tropism for macrophage cells, leading to a deadly disease called feline infectious peritonitis (FIP). In this study, monocyte-derived macrophages were isolated by CD14-positive selection in venous whole blood from 26 cats with FIP and 32 FCoV-positive healthy cats. Phagocytosis and respiratory burst activities were investigated and compared between the groups. This is the first study comparing macrophage activity in cats affected by FIP and healthy cats positive for FCoV infection. Our results showed that in cats with FIP, the phagocytic and respiratory burst activities were significantly lower. Our results support the possible role of host immunity in Coronaviridae pathogenesis in cats, supporting future research on the immune defense against this systemic disease.

MRGPRX2 antagonist GE1111 attenuated DNFB-induced atopic dermatitis in mice by reducing inflammatory cytokines and restoring skin integrity.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterised by itching, erythema, and epidermal barrier dysfunction. The pathogenesis of AD is complex and multifactorial; however,mast cell (MC) activation has been reported to be one of the crucial mechanisms in the pathogenesis of AD. The MC receptor Mas related G protein-coupled receptor-X2 (MRGPRX2) has been identified as a prominent alternative receptor to the IgE receptor in causing MC activation and the subsequent release of inflammatory mediators. The current study aimed to evaluate the therapeutic effect of a novel small molecule MRGPRX2 antagonist GE1111 in AD using in vitro and in vivo approaches. We developed an in vitro cell culture disease model by using LAD-2 MC, HaCaT keratinocytes and RAW 264.7 macrophage cell lines. We challenged keratinocytes and macrophage cells with CST-14 treated MC supernatant in the presence and absence of GE1111 and measured the expression of tight junction protein claudin 1, inflammatory cytokines and macrophage phagocytosis activity through immunohistochemistry, western blotting, RT-qPCR and fluorescence imaging techniques. In addition to this, we developed a DFNB-induced AD model in mice and evaluated the protective effect and underlying mechanism of GE1111. Our in vitro findings demonstrated a potential therapeutic effect of GE1111, which inhibits the expression of TSLP, IL-13, MCP-1, TNF-a, and IL-1ß in MC and keratinocytes. In addition to this, GE1111 was able to preserve the expression of claudin 1 in keratinocytes and the phagocytotic activity of macrophage cells. The in vivo results demonstrated that GE1111 treatment significantly reduced phenotypic changes associated with AD (skin thickening, scaling, erythema and epidermal thickness). Furthermore, immunohistochemical analysis demonstrated that GE1111 treatment preserved the expression of the tight junction protein Involucrin and reduced the expression of the inflammatory mediator periostin in the mouse model of AD. These findings were supported by gene and protein expression analysis, where GE1111 treatment reduced the expression of TSLP, IL-13, and IL-1ß, as well as downstream signalling pathways of MRGPRX2 in AD skin lesions. In conclusion, our findings provide compelling in vitro and in vivo evidence supporting the contribution of MRGPRX2-MC interaction with keratinocytes and macrophages in the pathogenesis of AD.

TREM2 protects against inflammation by regulating the release of mito-DAMPs from hepatocytes during liver fibrosis

BackgroundLiver fibrosis typically develops as a result of chronic liver injury, which involves inflammatory and regenerative processes. The triggering receptor expressed on myeloid cells 2 (TREM2), predominantly expressing in hepatic non-parenchymal cells, plays a crucial role in regulating the function of macrophages. However, its mechanism in liver fibrosis remains poorly defined. MethodsExperimental liver fibrosis models in wild type and TREM2−/− mice, and in vitro studies with AML-12 cells and Raw264.7 cells were conducted. The expression of TREM2 and related molecular mechanism were evaluated by using samples from patients with liver fibrosis. ResultsWe demonstrated that TREM2 was upregulated in murine model with liver fibrosis. Mice lacking TREM2 exhibited reduced phagocytosis activity in macrophages following carbon tetrachloride (CCl4) intoxication. As a result, there was an increased accumulation of necrotic apoptotic hepatocytes. Additionally, TREM2 knockout aggravated the release of mitochondrial damage-associated molecular patterns (mito-DAMPs) from dead hepatocytes during CCl4 exposure, and further promoted the occurrence of macrophage-mediated M1 polarization. Then, TREM2−/− mice showed more serious fibrosis pathological changes. In vitro, the necrotic apoptosis inhibitor GSK872 effectively alleviated the release of mito-DAMPs in AML-12 cells after CCl4 intoxication, which confirmed that mito-DAMPs originated from dead liver cells. Moreover, direct stimulation of Raw264.7 cells by mito-DAMPs from liver tissue can induce intracellular inflammatory response. More importantly, TREM2 was elevated and inflammatory factors were markedly accumulated surrounding dead cells in the livers of human patients with liver fibrosis. ConclusionOur study highlights that TREM2 serves as a negative regulator of liver fibrosis, suggesting its potential as a novel therapeutic target.

Mechanisms of ingested polystyrene micro-nanoplastics (MNPs) uptake and translocation in an in vitro tri-culture small intestinal epithelium

Micro and nanoplastics (MNPs) are now ubiquitous contaminants of food and water. Many cellular and animal studies have shown that ingested MNPs can breach the intestinal barrier to reach the circulation. To date however, the cellular mechanisms involved in intestinal absorption of MNPs have not been investigated with physiologically relevant models, and thus remain unknown. We employed in vitro simulated digestion, a tri-culture small intestinal epithelium model, and a panel of inhibitors to assess the contributions of the possible mechanisms to absorption of 26 nm carboxylated polystyrene (PS26C) MNPs. Inhibition of ATP synthesis reduced translocation by only 35 %, suggesting uptake by both active endocytic pathways and passive diffusion. Translocation was also decreased by inhibition of dynamin and clathrin, suggesting involvement of clathrin mediated endocytosis (CME) and fast endophilin-mediated endocytosis (FEME). Inhibition of actin polymerization also significantly reduced translocation, suggesting involvement of macropinocytosis or phagocytosis. However, inhibition of the Na+-H+ exchanger had no effect on translocation, thus ruling out macropinocytosis. Together these results suggest uptake by passive diffusion as well as by active phagocytosis, CME, and FEME pathways. Further studies are needed to assess uptake mechanisms for other environmentally relevant MNPs as a function of polymer, surface chemistry, and size.

Analysis of Lysine Metabolism in a Rat Model of Type 2 Diabetes Reveals Modulation of Endocytosis and Glucose Metabolism

Lysine is an essential amino acid that serves as a building block for proteins and plays a role in metabolic processes. While a recent study provided evidence that lysine is beneficial for hypertension-associated kidney disease, possibly through inhibiting renal tubular reabsorption and forming new lysine conjugates, knowledge about the role of lysine in diabetic nephropathy (DN) is rudimentary. Therefore, the aim of the study was to investigate the role of lysine in kidney function in the setting of DN and its underlying mechanisms. We hypothesized that lysine may impact the kidneys in the following ways: 1) by mediating protein endocytosis in proximal tubules; 2) by altering protein abundance in the kidneys and other organs; and 3) by disrupting metabolic pathways through the formation of conjugates or degradation products. We investigated the effects of lysine on proximal tubule endocytosis and other organs using proteomic approaches and studied changes in lysine conjugates and metabolic products using targeted metabolomics. The results of urinary proteomics suggested that lysine’s impact on endocytosis may be associated with the inhibition of megalin and cubilin. Furthermore, lysine affected proteins related to glucose metabolism. Subsequently, we proceeded to investigate the influence of lysine on various organs in Type 2 Diabetic Nephropathy (T2DN) rats. Gene Ontology (GO) enrichment revealed that lysine exerts tissue-specific effects on different organs, with pathways enriched in the kidney including phagocytosis and enzyme inhibitor activity. Additionally, within the kidney, the lysine-treated group showed downregulation of glycolysis-related enzymes. The metabolomics results showed that lysine leads to a decrease in glucose and an increase in fructose 1,6-bisphosphate, while also resulting in an upregulation of related metabolites in the tricarboxylic acid cycle (TCA cycle). Our findings showed changes in the protein composition involved in lysine-mediated endocytosis and the impact of lysine on glucose metabolism enzymes and metabolites. The specific molecular mechanisms are still under investigation. R01 DK135644 and I01 BX004024 (to AS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Highly Motile Neutrophil Cytoplast ameliorate Bacterial Pneumonia

Abstract Polymorphonuclear neutrophils (PMN) function as innate immune cell through rapid mobilization at site of infection and prompt phagocytosis and bactericidal activity. We addressed in vivo in mouse lungs the function of cytoplasts (PMNcyt), the PMN generated following release of extracellular traps (NETs). We found ~50% of PMN lost their nucleus post-NETosis following pneumonia induced by Pseudomonas aeruginosa (PA), and become PMNcyt. Interestingly, , PMNcyt in lung microvessels showed &amp;gt;2 greater velocity and &amp;gt; 3 greater deformability as compared to PMN. PMNcyt were also 9x more efficient in killing airway PA as compared to PMN. Using the protein arginine deiminase 4 (PAD4) inhibitor (Cl-amidine) to prevent NET formation in mice, we prevented the generation of PMNcyt demonstrating that PMNcyt were derived post-vital NETosis. We also showed PMNcyt retained the ability to phagocytose PA. Moreover, the inhibition of PMNcyt generation by PAD4 blocker induced severe lung injury in the pneumonia model, indicating an important anti-bacterial protective role of PMNcyt. Adoptive transfer of PMNcyt in PA infected mice also significantly reduced bacterial load and prevented lung tissue injury in mice. The results suggest a crucial host-defense function of PMNcyt generated post vital NETosis and their usefulness as cell-based therapy in inflammatory diseases such as pneumonia.

In Vitro Screening of Lactic Acid Bacteria with RAW264.7 Macrophages and the Immunoregulatory Mechanism

Lactic acid bacteria (LAB) are commonly consumed as probiotics to improve gut barrier function and boost the immune system. This study aimed to screen LAB with high immunomodulatory activity using RAW264.7 macrophages. According to the results, Limosilactobacillus reuteri AUc2301 was selected from 84 screened strains that can stimulate RAW264.7 cell proliferation. Limosilactobacillus reuteri AUc2301 significantly enhanced the phagocytosis activity of RAW264.7 cells. In the ELISA test, Limosilactobacillus reuteri AUc2301 significantly promoted the release of interleukin-6, IL-1β, the tumor necrosis factor, and nitric oxide in RAW264.7 macrophages. In addition, Limosilactobacillus reuteri AUc2301 significantly inhibited the excessive release of IL-6, IL-1β, TNF-α, prostaglandin E2 as well as NO and the high expression of cyclooxygenase-2 in RAW264.7 macrophages induced by lipopolysaccharide. In further mechanism studies, Limosilactobacillus reuteri AUc2301 could regulate the nuclear factor-κB signaling pathway in RAW264.7 macrophages. Collectively, the screened Limosilactobacillus reuteri AUc2301 showed good immunomodulatory activity in vitro, and it has the potential to be developed as a novel probiotic.