Phagocytic Capacity Research Articles

Activation of leukotriene B4 receptor 1 is a prerequisite for complement receptor 3-mediated antifungal responses of neutrophils.

Invasive fungal infections are life-threatening, and neutrophils are vital cells of the innate immune system that defend against them. The role of LTA4H-LTB4-BLT1 axis in regulation of neutrophil responses to fungal infection remains poorly understood. Here, we demonstrated that the LTA4H-LTB4-BLT1 axis protects the host against Candida albicans and Aspergillus fumigatus, but not Cryptococcus neoformans infection, by regulating the antifungal activity of neutrophils. Our results show that deleting Lta4h or Blt1 substantially impairs the fungal-specific phagocytic capacity of neutrophils. Moreover, defective activation of the spleen tyrosine kinase (Syk) and extracellular signal-related kinase (ERK1/2) pathways in neutrophils accompanies this impairment. Mechanistically, BLT1 regulates CR3-mediated, β-1,3-glucan-induced neutrophil phagocytosis, while a physical interaction with CR3 with slight influence on its dynamics is observed. Our findings thus demonstrate that the LTA4H-LTB4-BLT1 axis is essential for the phagocytic function of neutrophils in host antifungal immune response against Candida albicans and Aspergillus fumigatus.

Myeloid-specific knockout of Notch-1 inhibits MyD88- and TRIF-mediated TLR signaling pathways by regulating oxidative stress-SHP2 axis, thus restraining aneurysm progression.

Notch-1 is a signal regulatory protein with extensive effects in myeloid cells, but its role in aneurysms remains to be fully clarified. In this study, therefore, the aneurysm mouse model with myeloid-specific knockout of Notch-1 was established to observe the role of Notch-1 in aneurysm progression. The effect of Notch-1 was assessed by pathological staining and Western blotting. It was found that after myeloid-specific knockout of Notch-1 in the aneurysm mouse model, the area of aneurysms and the macrophage infiltration were significantly reduced, the damage to arterial elastic plates was significantly relieved, and the oxidative stress level significantly declined. The results of Western blotting showed that after myeloid-specific knockout of Notch-1, the levels of oxidative stress-related proteins p22 and p47 in aneurysm tissues significantly declined, accompanied by a significant increase in the protein level of Src homology 2 domain-containing tyrosine phosphatase-2 (SHP2). In addition, the levels of phosphorylated myeloid differential protein-88 (MyD88), TIR domain-containing adaptor-inducing interferon-β (TRIF) and nuclear factor-κB (NF-κB), and inflammatory cytokines interferon-γ (IFN-γ), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) also significantly decreased after myeloid-specific knockout of Notch-1. Following myeloid-specific knockout of Notch-1, the phagocytic capacity of macrophages was enhanced by promoting the SHP2 signaling pathway. Notch-1 in monocytes/macrophages can activate the Toll-like receptor (TLR)-mediated inflammatory and stress responses by activating oxidative stress and inhibiting the SHP2 protein expression, thus facilitating aneurysm progression.

Bystander activation of microglia by Brucella abortus-infected astrocytes induces neuronal death via IL-6 trans-signaling.

Inflammation plays a key role in the pathogenesis of neurobrucellosis where glial cell interactions are at the root of this pathological condition. In this study, we present evidence indicating that soluble factors secreted by Brucella abortus-infected astrocytes activate microglia to induce neuronal death. Culture supernatants (SN) from B. abortus-infected astrocytes induce the release of pro-inflammatory mediators and the increase of the microglial phagocytic capacity, which are two key features in the execution of live neurons by primary phagocytosis, a recently described mechanism whereby B. abortus-activated microglia kills neurons by phagocytosing them. IL-6 neutralization completely abrogates neuronal loss. IL-6 is solely involved in increasing the phagocytic capacity of activated microglia as induced by SN from B. abortus-infected astrocytes and does not participate in their inflammatory activation. Both autocrine microglia-derived and paracrine astrocyte-secreted IL-6 endow microglial cells with up-regulated phagocytic capacity that allows them to phagocytose neurons. Blocking of IL-6 signaling by soluble gp130 abrogates microglial phagocytosis and concomitant neuronal death, indicating that IL-6 activates microglia via trans-signaling. Altogether, these results demonstrate that soluble factors secreted by B. abortus-infected astrocytes activate microglia to induce, via IL-6 trans-signaling, the death of neurons. IL-6 signaling inhibition may thus be considered a strategy to control inflammation and CNS damage in neurobrucellosis.

Tissue factor overexpression promotes resistance to KRAS-G12C inhibition in non-small cell lung cancer

The recently approved KRASG12C mutation-specific inhibitors sotorasib and adagrasib (KRASG12C-I) represent a promising therapy for KRASG12C-driven non-small cell lung cancer (NSCLC). However, many eligible patients do not benefit due to intrinsic or acquired drug resistance. Tissue factor (TF) is overexpressed in KRAS-mutated (KRASmut) NSCLC and is the target of the FDA-approved ADC Tivdak. Here, we employed HuSC1-39, the parent antibody of a clinical stage TF-ADC (NCT04843709), to investigate the role of TF in KRASmut NSCLC. We found that patients with TF-overexpression had poor survival, elevated P-ERK/P-AKT activity levels and low immune effector cell infiltration in the tumor. In a panel of KRASG12C cell lines, KRASG12C-I response correlated with suppression of TF mRNA, which was not observed in resistant cells. In the drug resistant cells, TF-overexpression relied on an mTORC2-mediated and proteasome-dependent pathway. Combination treatment of HuSC1-39 or mTORC1/2 inhibitor MTI-31 with KRASG12C-I each produced synergistic antitumor efficacy in cell culture and in an orthotopic lung tumor model. TF-depletion in the resistant cells diminished epithelial mesenchymal transition, reduced tumor growth and greatly sensitized KRASG12C-I response. Moreover, employing immunohistochemistry and coculture studies, we demonstrated that HuSC1-39 or MTI-31 reset the tumor microenvironment and restore KRASG12C-I sensitivity by reshaping an M1-like macrophage profile with greatly enhanced phagocytic capacity toward tumor cell killing. Thus, we have identified the TF/mTORC2 axis as a critical new mechanism for triggering immunosuppression and KRASG12C-I resistance. We propose that targeting this axis with HuSC1-39 or MTI-31 will improve KRASG12C-I response in KRAS-driven NSCLC.

Controling the cytoskeleton during CEACAM3-mediated phagocytosis

Phagocytosis, an innate defense mechanism of multicellular animals, is initiated by specialized surface receptors. A phagocytic receptor expressed by human polymorphonuclear granulocytes, the major professional phagocytes in our body, is one of the fastest evolving human proteins implying a special role in human biology. This receptor, CEACAM3, is a member of the CarcinoEmbryonic Antigen-related Cell Adhesion Molecule (CEACAM) family and dedicated to the immediate recognition and rapid internalization of human-restricted pathogens. In this focused contribution, we will review the special adaptations of this protein, which co-evolves with different species of mucosa-colonizing bacteria. While the extracellular Immunoglobulin-variable (IgV)-like domain recognizes various bacterial adhesins, an Immunoreceptor Tyrosine-based Activation Motif (ITAM)-like sequence in the cytoplasmic tail of CEACAM3 constitutes the central signaling hub to trigger actin rearrangements needed for efficient phagocytosis. A major emphasis of this review will be placed on recent findings, which have revealed the multi-level control of this powerful phagocytic device. As tyrosine phosphorylation and small GTPase activity are central for CEACAM3-mediated phagocytosis, the counterregulation of CEACAM3 activity involves the receptor-type protein tyrosine phosphatase J (PTPRJ) as well as the Rac-GTP scavenging protein Cyri-B. Interference with such negative regulatory circuits has revealed that CEACAM3-mediated phagocytosis can be strongly enhanced. In principle, the knowledge gained by studying CEACAM3 can be applied to other phagocytic systems and opens the door to treatments, which boost the phagocytic capacity of professional phagocytes.

IPSC-derived PSEN2 (N141I) astrocytes and microglia exhibit a primed inflammatory phenotype

BackgroundWidescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer’s disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells.MethodsiPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aβ42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aβ42.ResultsAD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100β and increased secretion and phagocytosis of Aβ42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia.ConclusionOur study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a ‘primed’ phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aβ42 production and phagocytosis.

Stability of shark (Centrophorus sp.) liver oil rich in squalene as an immunostimulant

Shark liver oil has been widely produced in Indonesia; however, many shark liver oil products still do not meet the established standards. This research involves the addition of antioxidants to stabilize the oil as an effort to extend its shelf life. The objectives of this study were to determine the immunostimulant activity, assessed the synergistic effect of vitamin E in shark liver oil rich in squalene, and identified the optimum concentration of the antioxidant vitamin E in maintaining its stability. Immunostimulant activity was tested by observing spleenocyte proliferation, phagocytic index, and capacity in the peritoneal cavity cells of mice. The stability of the oil was examined by an accelerated method using an oven, what was called the schaal oven test at 40 °C. The research results indicate that shark liver oil possesses immunostimulant effects; however, with an increasing dosage of fish oil, there is no significant improvement. The presence of vitamin E in cod liver oil exhibited a synergistic effect on spleenocyte proliferation for all dosages, while the phagocytic index and capacity at a dosage of 100 μg/mL showed synergy. However, at a dosage of 50 μg/mL, the phagocytic index and capacity did not exhibit synergy with the immunostimulant. The optimal concentration of the antioxidant vitamin E in maintaining oil stability was found to be 100 ppm.

Flow cytometry-assisted analysis of phenotypic maturation markers on an immortalized dendritic cell line.

Dendritic cells (DCs), and especially so conventional type I DCs (cDC1s), are fundamental regulators of anticancer immunity, largely reflecting their superior ability to engulf tumor-derived material and process it for cross-presentation on MHC Class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). Thus, investigating key DC functions including (but not limited to) phagocytic capacity, expression of CTL-activating ligands on the cell surface, and cross-presentation efficacy is an important component of multiple immuno-oncology studies. Unfortunately, DCs are terminally differentiated cells, implying that they cannot be propagated indefinitely in vitro and hence must be generated ad hoc from circulating or bone marrow-derived precursors, which presents several limitations. Here, we propose a simple, cytofluorometric method to quantify phenotypic activation markers including CD80, CD86 and MHC class II molecules on the surface of a conditionally immortalized immature DC line that can be indefinitely propagated in vitro but also driven into maturation at will with a simple change in culture conditions. Upon appropriate scaling and automatization, this approach is compatible with high-throughput screening programs for the discovery of novel DC activators that do not suffer from batch variability and other limitations associated with the generation of fresh DCs.

Taurine influences the phagocytic function of retinal pigment epithelium cells in healthy and dystrophic animals

Aims/Purpose: To investigate the influence of taurine on the phagocytic function of the retinal pigment epithelium (RPE) in normal and dystrophic rats.Methods: Normal albino Sprague–Dawley (SD) and pigmented dystrophic Royal College of Surgeons (RCS‐p+) rats that suffer an inherited photoreceptor degeneration due to RPE phagocytosis impairment were used for the study. Taurine depletion was induced in SD rats (n = 8) by providing β‐alanine in drinking water (3%) from postnatal day 21 (P21). Taurine supplementation was administered to RCS‐p+ rats (n = 8) in drinking water (0.2 M) from P21. Untreated RCSp+ (n = 8) and SD (n = 8) rats served as control groups. Rats were processed at P81 (SD) and at P45 (RCS) and received an intravitreal injection (IV) of. 1.5 μL of Fluorogold (FG, 3%) diluted in saline 24 h before processing. The RPE was dissected, flat‐mounted and immunodetected with anti‐S opsin antibodies to label the RPE phagosomes which were quantified using ImageJ and size criteria.Results: The mean numbers (± SD) of phagosomes per RPE whole mount was 414 ± 118 in control SD rats, 1034 ± 528 in taurine‐depleted SD rats 88 ± 12 in untreated RCS and 119 ± 9 in taurine‐treated RCS rats. The numbers of phagosomes were significantly higher in SD rats than in RCS‐p+ rats (t‐test, p < 0.0001) documenting the impaired phagocytic function of RCS‐p+ rats. In SD rats, the number of phagosomes were significantly higher in taurine‐depleted rats evidencing that taurine depletion decreases the RPE phagocytic capacity (t‐test, p < 0.0059). Finally, in RCSp+ rats, the numbers of phagosomes were significantly higher in taurine‐treated than in non‐treated RCS rats (t‐test, p < 0.0001) indicating that taurine supplementation ameliorates the phagocytic capacity of the RPE cells in RCS‐p+ rats.Conclusions: Taurine influences the phagocytic capacity of the RPE cells in normal rats and in dystrophic RCS‐p+ rats.

The impact of macrophage phenotype and heterogeneity on the total internalized gold nanoparticle counts.

Macrophages play a pivotal role in the internalization and processing of administered nanoparticles (NPs). Furthermore, the phagocytic capacity and immunological properties of macrophages can vary depending on their microenvironment, exhibiting a spectrum of polarization states ranging from pro-inflammatory M1 to anti-inflammatory M2. However, previous research investigating this phenotype-dependent interaction with NPs has predominantly relied on semi-quantitative techniques or conventional metrics to assess intracellular NPs. Here, we focus on the interaction of human monocyte-derived macrophage phenotypes (M1-like and M2-like) with gold NPs (AuNPs) by combining population-based metrics and single-cell analysis by focused ion beam-scanning electron microscopy (FIB-SEM). The multimodal analysis revealed phenotype-dependent response and uptake behavior differences, becoming more pronounced after 48 hours. The study also highlighted phenotype-dependent cell-to-cell heterogeneity in AuNPs uptake and variability in particle number at the single-cell level, which was particularly evident in M2-like macrophages, which increases with time, indicating enhanced heteroscedasticity. Future efforts to design NPs targeting macrophages should consider the phenotypic variations and the distribution of NPs concentrations within a population, including the influence of cell-to-cell heterogeneity. This comprehensive understanding will be critical in developing safe and effective NPs to target different macrophage phenotypes.

Role of Phyllanthus niruri on the modulation of stress and immune responses in Nile tilapia, Oreochromis niloticus.

The use of nutraceuticals in aquaculture is getting attention to minimize oxidative stress and increase immunity of aquatic animals. In our experiment, we tested the potency of Phyllanthus niruri, as a stress-reducing and immune-stimulating agent in Nile tilapia, Oreochromis niloticus. We reared fish in a recirculating system for 90 days at low (5 g/L) and high (30 g/L) densities. We fed half of the low and high-density fish with commercial feed (control feed) and the other half, with 5% Phyllanthus niruri incorporated into commercial feed (supplemented feed). We assessed plasma cortisol, blood glucose, packed cell volume, plasma proteins, phagocytic capacity, respiratory burst activity, and lysozyme activity. None of these parameters showed any significant difference among the experimental groups. Based on our findings, we conclude that Phyllanthus niruri does not have any potential role in modulating stress and immune response in fish as a nutraceutical.

On the potential therapeutic role of taurine in retinal degenerative diseases

Taurine, considered a non‐essential amino acid that differs from other amino acids in that it contains a sulfonic acid instead of a carboxyl group, is the most abundant amino acid in the retina. Taurine plays a critical role in maintaining retinal health and the survival of retinal neurons, although its exact function in the retina is not yet fully understood. Interestingly, taurine shares structural similarities with β‐alanine, another non‐proteinogenic amino acid that can act as a competitive inhibitor of taurine receptors, allowing β‐alanine to be used to induce taurine depletion. β‐alanine induced taurine depletion results in the activation and migration of microglial cells in the retina, oxidative stress in the inner and outer nuclear and ganglion cell layers, loss of retinal synaptic connectivity, and impaired phagocytic capacity of retinal pigment epithelial cells. In addition, taurine depletion leads to photoreceptor and retinal ganglion cell loss. Most of these detrimental events are exacerbated by light exposure. These findings suggest that taurine levels are associated with retinal sensitivity to light.Given the important role of taurine in the retina, it has been proposed as a promising therapeutic candidate for retinal degenerative diseases. These diseases, which can be inherited or acquired, are a leading cause of irreversible blindness worldwide. Age‐related macular degeneration is the most common acquired cause of photoreceptor degeneration and a major public health concern due to the aging population. Retinitis pigmentosa is the most common inherited retinal degenerative disease, which is a heterogeneous group of sight‐threatening disorders. These diseases share photoreceptor degeneration as a common feature, typically due to genetic defects affecting the photoreceptors or the retinal pigment epithelium but can also be influenced by environmental factors. Additionally, inflammation and oxidative stress contribute to their development and progression. These diseases represent a major economic burden to healthcare systems and society. Therefore, it is imperative to develop preventive and/or therapeutic strategies that can impede or halt their progression.Taurine supplementation is a promising therapeutic approach for these diseases due to its antioxidant, anti‐apoptotic, and anti‐inflammatory properties. Our group has investigated the effects of taurine supplementation in the Royal College of Surgeons (RCS) rat, an established model of inherited retinal degeneration that manifests an impaired phagocytic capacity of the retinal pigment epithelium due to a MERKT gene mutation. Our findings demonstrate that taurine supplementation improves functional photoreceptor survival, decreases glial activation (including decreased GFAP expression in Müller cells and inhibition of microglial activation and migration), reduces oxidative stress in the outer and inner nuclear layers, suppresses apoptosis, and preserves synaptic connections in the RCS rat retina. Therefore, taurine is essential for retinal health and may provide neuroprotection against retinal degeneration thanks to its multifaceted properties.

Macrophage Dysfunction in Respiratory Disease.

Our understanding of the origin, phenotype, and function of pulmonary macrophages has evolved over recent years. The use of lineage tracing and single-cell RNA sequencing has led to a greater understanding of how these cells regulate homeostasis in the lung. The primary function of alveolar macrophages is to clear any inhaled particles or pathogens and they as well as tissue-resident cells also play a role in the clearance of apoptotic cells and the resolution of inflammation. Lung diseases affect over half a billion people globally and are attributable to 7% of all deaths each year. The common diseases are chronic obstructive pulmonary disease (COPD) and asthma but others that contribute to this statistic include cystic fibrosis and idiopathic pulmonary fibrosis (IPF). Macrophages are aberrant in all these diseases with a reduced phagocytic capacity and a high proinflammatory phenotype with changes to their capacity to resolve inflammation. The pathways leading to these macrophage dysfunctions differ with disease and may relate to the specific lung environment in each condition. However, there are clear changes in metabolic profiles and mitochondrial activity in many of these conditions that contribute to a change in macrophage phenotype towards a more proinflammatory, less homeostatic cell. Understanding the mechanisms that drive these changes will allow for more targeted therapies for the treatment of these long-term and debilitating conditions.

EYS mutation causes RPE dysfunction in a patient‐derived cellular model

Aims/Purpose: Although EYS‐associated retinal disease is normally described as a photoreceptor dysfunction, the natural role of the EYS protein is not yet clearly defined and it may also affect the RPE, as has been previously shown for other genes causing retinal disease. We have studied the impact of EYS gene mutations on the maturation and function of an RPE cellular model obtained from a patient.Methods: RPE was differentiated in vitro from a patient‐derived iPSC line and characterized. Morphological assessment of EYS‐RPE included light microscopy and electron microscopy. We studied gene expression by rt qPCR and transcriptomic analysis and cellular localization of the main RPE markers by immunofluorescence. For physiology, we tested the transepithelial resistance, phagocytic activity and secretion of PEDF and VEGF.Results: Microscopic phenotyping of the EYS‐RPE shows areas where cells grow in several layers, different from the healthy RPE model that grows as a monostratified epithelium. Moreover, the ultrastructure shows a loose attachment between cells and a disorganized distribution of the organelles and vesicles compared to the polarized distribution inside a healthy RPE cell, among other differences. Regarding gene expression, several RPE markers are downregulated in the EYS‐RPE pointing to a general dysregulation of the RPE metabolism and differentiation. The immunofluorescence study correlates with the gene expression data. As expected from the previous information, the EYS‐RPE physiology is affected, including a diminished phagocytic capacity and altered secretion of PEDF and VEGF.Conclusions: Our data indicate that EYS dysfunction is not only a photoreceptor disease but also affects RPE at many levels, from differentiation to impairing some of its key roles in the retina, which may contribute to retinal disease in EYS patients.

Blue mussel (Mytilus edulis) water extract ameliorates intestinal immune response in high-fat diet-streptozotocin-induced diabetic mice.

Diabetes mellitus is a metabolic disease characterized by high blood glucose levels or hyperglycemia. Diabetes causes a decrease in immune function in the human body. Mytilus edulis has been identified as having anti-inflammatory properties and the ability to improve inflammation. Thus, this study aimed to investigate the function of Matsu M. edulis water extract (MWE) in mediating the regulation of immune responses and dysregulating the intestinal immune system in hyperglycemia mouse models. The mice were treated with MWE for seven weeks. The results showed that treatment with MWE has the ability to decrease triglyceride and total cholesterol concentrations. MWE also increases the interleukin (IL)-10 concentration and natural killer cell activation. It also improves the phagocytic capacity of monocytes in the colon and the proliferative capacity of lymphocytes in the mesentery. Furthermore, MWE also regulates the IL-6 concentration and the ratio of T helper 17 cells to regulatory T cells. Collectively, this extract can improve dyslipidemia, inflammatory responses, and dysregulation of the intestinal immune system. Therefore, M. edulis water extract can be used as an alternative treatment to reduce diabetes complications.

Alpha-synuclein shapes monocyte and macrophage cell biology and functions by bridging alterations of autophagy and inflammatory pathways.

Introduction: Abnormal spreading of alpha-synuclein (αS), a hallmark of Parkinson's disease, is known to promote peripheral inflammation, which occurs in part via functional alterations in monocytes/macrophages. However, underlying intracellular mechanisms remain unclear. Methods: Herein we investigate the subcellular, molecular, and functional effects of excess αS in human THP-1 monocytic cell line, THP-1-derived macrophages, and at least preliminarily, in primary monocyte-derived macrophages (MDMs). In cells cultured w/wo recombinant αS (1μM) for 4h and 24h, by Confocal microscopy, Western Blot, RT-qPCR, Elisa, and Flow Cytometry we assessed: i) αS internalization; ii) cytokine/chemokine expression/secretion, and C-C motif chemokine receptor 2 (CCR2) levels; iii) autophagy (LC3II/I, LAMP1/LysoTracker, p62, pS6/total S6); and iv) lipid droplets (LDs) accumulation, and cholesterol pathway gene expression. Transwell migration assay was employed to measure THP-1 cell migration/chemotaxis, while FITC-IgG-bead assay was used to analyze phagocytic capacity, and the fate of phagocytosed cargo in THP-1-derived macrophages. Results: Extracellular αS was internalized by THP-1 cells, THP-1-derived macrophages, and MDMs. In THP1 cells, αS induced a general pro-inflammatory profile and conditioned media from αS-exposed THP-1 cells potently attracted unstimulated cells. However, CCL2 secretion peaked at 4h αS, consistent with early internalization of its receptor CCR2, while this was blunted at 24h αS exposure, when CCR2 recycled back to the plasma membrane. Again, 4h αS-exposed THP-1 cells showed increased spontaneous migration, while 24h αS-exposed cells showed reduced chemotaxis. This occurred in the absence of cell toxicity and was associated with upregulation of autophagy/lysosomal markers, suggesting a pro-survival/tolerance mechanism against stress-related inflammation. Instead, in THP-1-derived macrophages, αS time-dependently potentiated the intracellular accumulation, and release of pro-inflammatory mediators. This was accompanied by mild toxicity, reduced autophagy-lysosomal markers, defective LDs formation, as well as impaired phagocytosis, and the appearance of stagnant lysosomes engulfed with phagocytosed cargo, suggesting a status of macrophage exhaustion reminiscent of hypophagia. Discussion: In summary, despite an apparently similar pro-inflammatory phenotype, monocytes and macrophages respond differently to intracellular αS accumulation in terms of cell survival, metabolism, and functions. Our results suggest that in periphery, αS exerts cell- and context-specific biological effects bridging alterations of autophagy, lipid dynamics, and inflammatory pathways.

Rhynchophorus ferrugineus larvae: A novel source for combating broad-spectrum bacterial and fungal infections.

Antimicrobial resistance is a growing concern due to the growth of antibiotic-resistant microorganisms, which makes it difficult to treat infection. Due to its broad-spectrum antimicrobial properties against a diverse array of bacteria, both Gram-positive and Gram-negative bacteria, and fungi, Rhynchophorus ferrugineus larval antimicrobial peptides (AMPs) have demonstrated potential as antimicrobial agents for the treatment of microbial infections and prevention of antibiotic resistance. This study emphasizes the unexplored mechanisms of action of R. ferrugineus larvae against microorganisms. Among the most widely discussed mechanisms is the effect of AMPs in larvae in response to a threat or infection. Modulation of immune-related genes in the intestine and phagocytic capacity of its hemocytes may also affect the antimicrobial activity of R. ferrugineus larvae, with an increase in phenoloxidase activity possibly correlated with microbial clearance and survival rates of larvae. The safety and toxicity of R. ferrugineus larvae extracts, as well as their long-term efficacy, are also addressed in this paper. The implications of future research are explored in this paper, and it is certain that R. ferrugineus larvae have the potential to be developed as a broad-spectrum antimicrobial agent with proper investigation.

Increased phagocytosis capacity of circulating neutrophils in patients on continuous flow ventricular assist device support.

Neutrophils take part in the innate immune response, phagocytosis, and pro-inflammatory cytokine release. The phagocytic capacity of circulating neutrophils in patients on continuous flow (CF) ventricular assist device (VAD) has not been well studied. Blood samples from 14 patients undergoing CF-VAD implantation were collected and analyzed preoperatively (at baseline) and on postoperative days (POD) 3, 7, 14, and 28. Flow cytometry was used to assess the surface expression levels of CD62L, CD162, and macrophage antigen-1 (MAC-1) and neutrophil phagocytic capacity. Interleukin 1 (IL1), IL6, IL8, TNF-α, neutrophil elastase, and myeloperoxidase in plasma were measured using enzyme-linked immunosorbent assays. Among the 14 patients, seven patients had preoperative bridge device support. Relative to baseline, patients with no bridge device had elevated leukocyte count and neutrophil elastase by POD3 which normalized by POD7. Neutrophil activation level, IL6, IL8, and TNF-α increased by POD3 and sustained elevated levels for 7-14 days postoperatively. Elevated neutrophil phagocytic capacity persisted even until POD28. Similar patterns were observed in patients on a preoperative bridge device. Neutrophil activation and phagocytic capacity increased in response to VAD support, while inflammatory cytokines remain elevated for up to 2 weeks postoperatively. These findings may indicate that VAD implantation elicits circulating neutrophils to an abnormal preemptive phagocytotic phenotype.

Isolation and functional identification of immune cells in hemolymph of blood clams Tegillarca granosa

Blood clam Tegillarca granosa is a type of economically cultivated bivalve mollusk with red blood, and it primarily relies on hemocytes in its hemolymph for immune defense. However, there are currently no reports on the isolation and identification of immune cells in T. granosa, which hinders our understanding of their immune defense. In this study, we employed single-cell transcriptome sequencing (scRNA-seq) to visualize the molecular profile of hemocytes in T. granosa. Based on differential expression of immune genes and hemoglobin genes, hemocytes can be molecularly classified into immune cells and erythrocytes. In addition, we separated immune cells using density gradient centrifugation and demonstrated their stronger phagocytic capacity compared to erythrocytes, as well as higher levels of ROS and NO. In summary, our experiments involved the isolation and functional identification of immune cells in hemolymph of T. granosa. This study will provide valuable insights into the innate immune system of red-blood mollusks and further deepen the immunological research of mollusks.

Immune response of BV-2 microglial cells is impacted by peroxisomal beta-oxidation.

Microglia are crucial for brain homeostasis, and dysfunction of these cells is a key driver in most neurodegenerative diseases, including peroxisomal leukodystrophies. In X-linked adrenoleukodystrophy (X-ALD), a neuroinflammatory disorder, very long-chain fatty acid (VLCFA) accumulation due to impaired degradation within peroxisomes results in microglial defects, but the underlying mechanisms remain unclear. Using CRISPR/Cas9 gene editing of key genes in peroxisomal VLCFA breakdown (Abcd1, Abcd2, and Acox1), we recently established easily accessible microglial BV-2 cell models to study the impact of dysfunctional peroxisomal β-oxidation and revealed a disease-associated microglial-like signature in these cell lines. Transcriptomic analysis suggested consequences on the immune response. To clarify how impaired lipid degradation impacts the immune function of microglia, we here used RNA-sequencing and functional assays related to the immune response to compare wild-type and mutant BV-2 cell lines under basal conditions and upon pro-inflammatory lipopolysaccharide (LPS) activation. A majority of genes encoding proinflammatory cytokines, as well as genes involved in phagocytosis, antigen presentation, and co-stimulation of T lymphocytes, were found differentially overexpressed. The transcriptomic alterations were reflected by altered phagocytic capacity, inflammasome activation, increased release of inflammatory cytokines, including TNF, and upregulated response of T lymphocytes primed by mutant BV-2 cells presenting peptides. Together, the present study shows that peroxisomal β-oxidation defects resulting in lipid alterations, including VLCFA accumulation, directly reprogram the main cellular functions of microglia. The elucidation of this link between lipid metabolism and the immune response of microglia will help to better understand the pathogenesis of peroxisomal leukodystrophies.