Phagocytosis Ability Research Articles

Establishment of a Disease Model Using Patient-Specific Induced Pluripotent Stem Cells-Derived Trabecular Meshwork Cells in a Chinese Primary Open-Angle Glaucoma Mega-Pedigree.

Primary open-angle glaucoma (POAG) is one of the most common insidious blinding eye diseases. Understanding the pathogenic mechanisms of it is extremely important. It is accepted that POAG attacks specific ocular tissue, such as trabecular meshwork and optic nerve damage, which causes elevated intraocular pressure and optic nerve damage. This study aimed to develop a preliminary prediction model for this disease by establishing the patient-specific induced pluripotent stem cells (iPSCs)-derived trabecular meshwork cells (TMCs) (p-iPSCs-TMCs) in the largest POAG family named "GZ.1" in China and preliminarily analyze the pathogenic mechanisms. Peripheral blood of patients in GZ.1 and healthy individuals not belonging to the family were collected and reprogrammed into iPSCs. Then, the iPSCs were differentiated into iPSCs-TMCs. Next, their morphology and function were compared. Finally, their pathogenic mechanisms were analyzed. The patient-specific iPSCs (p-iPSCs) and healthy individual-specific iPSCs (n-iPSCs) were all successfully generated. Their morphology was quite similar to each other. However, p-iPSCs-TMCs exhibited compromised morphology and function. p-iPSCs-TMCs displayed the morphology of heterogeneous distribution and accumulation in clusters, while n-iPSCs-derived TMCs (n-iPSCs-TMCs) showed a uniformly distributed and homogenous appearance. Moreover, p-iPSCs-TMCs showed greater cell apoptosis (p < 0.01), impaired proliferating ability (24-h and 48-h time points: p < 0.05, 72-h and 96-h time points: p < 0.001), production of reactive oxygen species (p < 0.05), and impaired phagocytosis ability than n-iPSCs-TMCs (24-h, 48-h, and 72-h time points: p < 0.0001, 96-h time point: p < 0.001). The p-iPSCs-TMCs can be successfully differentiated from peripheral blood, while the cells show impaired morphology and function compared with n-iPSCs-TMCs. Given this, p-iPSCs-TMCs can serve as an ideal disease model for POAG in GZ.1. Our study on the morphology and function of iPSCs-TMCs in GZ.1 may provide a valuable tool for elucidating the pathogenesis of POAG.

Deletion of CD38 mitigates the severity of NEC in experimental settings by modulating macrophage-mediated inflammation

Necrotizing enterocolitis (NEC) is a form of potentially lethal gastrointestinal inflammation that primarily affects preterm neonates. It is crucial to recognize that, while the disease carries significant risks, timely and effective medical intervention can greatly enhance the chances of survival. Additionally, NEC is closely linked to the activation of macrophages, highlighting the complex interplay between the immune response and disease progression. CD38, acting as an ectoenzyme, catalyzes the hydrolysis of NAD+ to produce cyclic ADP-ribose (cADPR), a reaction critical for modulating cellular redox balance and energy homeostasis. This enzymatic activity is particularly pertinent in the context of necrotizing enterocolitis (NEC). In this research, we investigated whether CD38 deletion can elevate NAD+ levels to reduce macrophage-mediated inflammation and improve NEC severity. We show that NEC patients was associated with the increased CD38 expression in intestine and blood. These results were also observed in NEC mice, and CD38 deletion ameliorated NEC intestinal injury. Mechanistically, CD38 deletion elevated NAD+ levels that reduced oxidative stress and intestinal inflammation. Furthermore, CD38 deletion promoted M2 macrophage polarization, inhibited macrophage activation and phagocytosis ability. Thus, our results reveal a critical role for CD38 as an intracellular immune regulator for regulating macrophage activation and intestinal inflammation in NEC. Targeting CD38 and NAD+ signal maybe a promising strategy for treatment of NEC.

Reprogramming tumor-associated macrophages using exosomes from M1 macrophages

Macrophages, abundant in tumors, are classified as M1 or M2 types with M2 dominating the tumor microenvironment. Shifting macrophages from M2 to M1 using exosomes is a promising intervention. The properties of exosomes depend on their source cells. M1-exosomes are expected to polarize macrophages towards M1 phenotype. We compared M1-exosomes and M0-exosomes' effects on M2 macrophage polarization.The RAW264.7 cells were cultured and one group of them was exposed to LPS. The serum-free medium was collected and exosomes were extracted. Exosomes were analyzed by scanning and transmission electron microscopy, dynamic light scattering and Western blot. Subsequently, M1 or M0 exosomes were applied to M2 macrophages induced by IL4. The macrophages polarization, including M1 and M2 genes and surface markers expression, cytokines secretion, and phagocytosis ability were evaluated.It was demonstrated that M1-exosomes induced macrophage polarization toward the M1 phenotype, characterized by an upregulation of M1-specific markers and a downregulation of M2 markers. Furthermore, the secretion of TNF-α was increased, while the secretion of IL-10 was decreased. The phagocytosis ability of M1-exosome-treated macrophages was also augmented.This research suggested that M1-exosomes might be promising candidates for modulating immune response in situations marked by an overabundance of M2 polarization, like in cancer.

Bactericidal ability of target acidic phospholipids and phagocytosis of CDC42 GTPase-mediated cytoskeletal rearrangement underlie functional conservation of CXCL12 in vertebrates.

Chemokine CXCL12 plays a crucial role in both direct bactericidal activity and phagocytosis in humans. However, the mechanisms and evolutionary functions of these processes in vertebrates remain largely unknown. In this study, we found that the direct bactericidal activity of CXCL12 is highly conserved across various vertebrate lineages, including Arctic lamprey (Lampetra japonica), Basking shark (Cetorhinus maximus), grass carp (Ctenopharyngodon idella), Western clawed frog (Xenopus tropicalis), Green anole (Anolis carolinensis), chicken (Gallus gallus), and human (Homo sapiens). CXCL12 also has been shown to promote phagocytosis in lower and higher vertebrates. We then employed C. idella CXCL12a (CiCXCL12a) as a model to further investigate its immune functions and underlying mechanisms. CiCXCL12a exerts direct broad-spectrum antibacterial activity by targeting bacterial acidic phospholipids, resulting in bacterial cell membrane perforation, and eventual lysis. Monocytes/macrophages are attracted to the infection sites for phagocytosis through the rapid production of CiCXCL12a during bacterial infection. CiCXCL12a induces CDC42 and CDC42 GTPase activation, which in turn mediates F-actin polymerization and cytoskeletal rearrangement. The interaction between F-actin and Aeromonas hydrophila facilitates bacterial internalization into monocytes/macrophages. Additionally, A. hydrophila is colocalized within early endosomes, late endosomes and lysosomes, ultimately degrading within phagolysosomes. CiCXCL12a also activates PI3K-AKT, JAK-STAT5 and MAPK-ERK signaling pathways. Notably, only the PI3K-AKT signaling pathway inhibits LPS-induced monocyte/macrophage apoptosis. Thus, CiCXCL12a plays key roles in reducing tissue bacterial loads, attenuating organ injury, and decreasing mortality rates. Altogether, our findings elucidate the conserved mechanisms underlying CXCL12-mediated bactericidal activity and phagocytosis, providing novel perspectives into the immune functions of CXCL12 in vertebrates.

Seminal plasma inhibits Chlamydia trachomatis infection in vitro, and may have consequences on mucosal immunity

Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female.

Lipids Extracted from Aptocyclus ventricosus Eggs Possess Immunoregulatory Effects on RAW264.7 Cells by Activating the MAPK and NF-κB Signaling Pathways.

This study was conducted to evaluate the potential anti-inflammatory and immune-enhancement properties of lipids derived from Aptocyclus ventricosus eggs on RAW264.7 cells. Firstly, we determined the fatty acid compositions of A. ventricosus lipids by performing gas chromatography analysis. The results showed that A. ventricosus lipids contained saturated fatty acids (24.37%), monounsaturated fatty acids (20.90%), and polyunsaturated fatty acids (54.73%). They also contained notably high levels of DHA (25.91%) and EPA (22.05%) among the total fatty acids. Our results for the immune-associated biomarkers showed that A. ventricosus lipids had immune-enhancing effects on RAW264.7 cells. At the maximum dose of 300 µg/mL, A. ventricosus lipids generated NO (119.53%) and showed greater phagocytosis (63.69%) ability as compared with untreated cells. A. ventricosus lipids also upregulated the expression of iNOS, IL-1β, IL-6, and TNF-α genes and effectively upregulated the phosphorylation of MAPK (JNK, p38, and ERK) and NF-κB p65, indicating that these lipids could activate the MAPK and NF-κB pathways to stimulate macrophages in the immune system. Besides their immune-enhancing abilities, A. ventricosus lipids significantly inhibited LPS-induced RAW264.7 inflammatory responses via the NF-κB and MAPK pathways. The results indicated that these lipids significantly reduced LPS-induced NO production, showing a decrease from 86.95% to 38.89%. Additionally, these lipids downregulated the expression of genes associated with the immune response and strongly suppressed the CD86 molecule on the cell surface, which reduced from 39.25% to 33.80%. Collectively, these findings imply that lipids extracted from A. ventricosus eggs might have biological immunoregulatory effects. Thus, they might be considered promising immunomodulatory drugs and functional foods.

Maresin2 negatively regulates DC’s maturation via the MAPK/NF-κB pathway in DCs

ObjectiveTo observe the effects and mechanisms of Maresin2 on the function of DCs(Dendritic cells). MethodThe levels of IL-6, IL-12, TNF-α and IL-1β secreted by BMDCs (Bone marrow-derived Dendritic cells) after Maresin2 treatment were detected by ELISA. At the same time, the expressions of costimulatory molecules CD40 and CD86 on the surface, the ability of phagocytosis of ovalbumin(OVA) antigen, and antigen presentation function in BMDCs were analyzed by flow cytometry. Finally, MAPK and NF-κB pathway signaling phosphorylation in Maresin2-treated BMDCs were detected by western blot. ResultsThe secretion levels of IL-6, IL-12, TNF-α and IL-1β were significantly decreased in the Maresin2 treatment group after LPS treatment (P < 0.05). The expression levels of CD86 and CD40 were significantly decreased after Maresin2 treatment (P < 0.05). Maresin2 enhanced the phagocytosis ability of ovalbumin(OVA) (P < 0.05), but the ability of antigen presentation of BMDCs with the treatment of Maresin2 changed slightly (P > 0.05). Phosphorylation of p38, JNK, p65, ikka/β and ERK peaked at 15 min in the LPS group, while phosphorylation of p-p38 and p-ERK weakened 30 min and 60 min after treatment with Maresin2. ConclusionsMaresin2 inhibits inflammatory cytokine secretion but enhances phagocytosis via the MAPK/NF-κB pathway in BMDCs, which may contribute to negatively regulating inflammation.

Dual Template Molecularly Imprinted Polymers Targeting Blockade of CD47 for Enhanced Macrophage Phagocytosis and Synergistic Antimetabolic Therapy.

Glycinamide ribonucleotide formyltransferase (GARFT) is an important enzyme in the folate metabolism pathway, and chemical drugs targeting GARFT have been used in tumor treatments over the past few decades. The development of novel antimetabolism drugs that target GARFT with improved performance and superior activity remains an attractive strategy. Herein, we proposed a targeted double-template molecularly imprinted polymer (MIP) for enhancing macrophage phagocytosis and synergistic antimetabolic therapy. The double-template MIP was prepared by imprinting the exposed peptide segment of the extracellular domain of CD47 and the active center of GARFT. Owing to the imprinted cavities on the surface of MIP, it can actively target cancer cells and mask the "do not eat me" signal upon binding to CD47 thereby blocking the CD47-SIRPα pathway and ultimately enhancing phagocytosis by macrophages. In addition, MIP can specifically bind to the active center of GARFT upon entry into the cells, thereby inhibiting its catalytic activity and ultimately interfering with the normal expression of DNA. A series of cell experiments demonstrated that MIP can effectively target CD47 overexpressed 4T1 cancer cells and inhibit the growth of 4T1 cells. The enhanced phagocytosis ability of macrophages-RAW264.7 cells was also clearly observed by confocal imaging experiments. In vivo experiments also showed that the MIP exhibited a satisfactory tumor inhibition effect. Therefore, this study provides a new idea for the application of molecular imprinting technology to antimetabolic therapy in conjunction with macrophage-mediated immunotherapy.

SHISA3 Reprograms Tumor-Associated Macrophages Toward an Antitumoral Phenotype and Enhances Cancer Immunotherapy.

The main challenge for immune checkpoint blockade (ICB) therapy lies in immunosuppressive tumor microenvironment (TME). Repolarizing M2-like tumor-associated macrophages (TAMs) into inflammatory M1 phenotype is a promising strategy for cancer immunotherapy. Here, this study shows that the tumor suppressive protein SHISA3 regulates the antitumor functions of TAMs. Local delivery of mRNA encoding Shisa3 enables cancer immunotherapy by reprogramming TAMs toward an antitumoral phenotype, thus enhancing the efficacy of programmed cell death 1 (PD-1) antibody. Enforced expression of Shisa3 in TAMs increases their phagocytosis and antigen presentation abilities and promotes CD8+ T cell-mediated antitumor immunity. The expression of SHISA3 is induced by damage/pathogen-associated molecular patterns (DAMPs/PAMPs) in macrophages via nuclear factor-κB (NF-κB) transcription factors. Reciprocally, SHISA3 forms a complex with heat shock protein family A member 8 (HSPA8) to activate NF-κB signaling thus maintaining M1 polarization of macrophages. Knockout Shisa3 largely abolishes the antitumor efficacy of combination immunotherapy with Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) and PD-1 antibody. It further found that higher expression of SHISA3 in antitumoral TAMs is associated with better overall survival in lung cancer patients. Taken together, the findings describe the role of SHISA3 in reprogramming TAMs that ameliorate cancer immunotherapy.

Effects of Fucoidans on Activated Retinal Microglia.

Sulfated marine polysaccharides, so-called fucoidans, have been shown to exhibit anti-inflammatory and immunomodulatory activities in retinal pigment epithelium (RPE). In this study, we tested the effects of different fucoidans (and of fucoidan-treated RPE cells) on retinal microglia to investigate whether its anti-inflammatory effect can be extrapolated to the innate immune cells of the retina. In addition, we tested whether fucoidan treatment influenced the anti-inflammatory effect of RPE cells on retinal microglia. Three fucoidans were tested (FVs from Fucus vesiculosus, Fuc1 and FucBB04 from Laminaria hyperborea) as well as the supernatant of primary porcine RPE treated with fucoidans for their effects on inflammatory activated (using lipopolysaccharide, LPS) microglia cell line SIM-A9 and primary porcine retinal microglia. Cell viability was detected with a tetrazolium assay (MTT), and morphology by Coomassie staining. Secretion of tumor necrosis factor alpha (TNFα), interleukin 1 beta (IL1β) and interleukin 8 (IL8) was detected with ELISA, gene expression (NOS2 (Nitric oxide synthase 2), and CXCL8 (IL8)) with qPCR. Phagocytosis was detected with a fluorescence assay. FucBB04 and FVs slightly reduced the viability of SIM-A9 and primary microglia, respectively. Treatment with RPE supernatants increased the viability of LPS-treated primary microglia. FVs and FucBB04 reduced the size of LPS-activated primary microglia, indicating an anti-inflammatory phenotype. RPE supernatant reduced the size of LPS-activated SIM-A9 cells. Proinflammatory cytokine secretion and gene expression in SIM-A9, as well as primary microglia, were not significantly affected by fucoidans, but RPE supernatants reduced the secretion of LPS-induced proinflammatory cytokine secretion in SIM-A9 and primary microglia. The phagocytosis ability of primary microglia was reduced by FucBB04. In conclusion, fucoidans exhibited only modest effects on inflammatorily activated microglia by maintaining their cell size under stimulation, while the anti-inflammatory effect of RPE cells on microglia irrespective of fucoidan treatment could be confirmed, stressing the role of RPE in regulating innate immunity in the retina.

Structural characteristics, immune-activating mechanisms in vitro, and immunomodulatory effects in vivo of the exopolysaccharide EPS53 from Streptococcus thermophilus XJ53

Our previous investigations have successfully identified the repeating structural units of EPS53, an exopolysaccharide derived from Streptococcus thermophilus XJ53 fermented milk, and substantiated its potential immunomodulatory properties. The present study further elucidated the structural characteristics of EPS53 and investigated the underlying mechanisms governing its in vitro immunoreactivity as well as its in vivo immunoreactivity. The results obtained from multi-detector high performance gel filtration chromatography revealed that EPS53 adopted a rigid rod conformation in aqueous solution, with the weight-average molecular weight of 1464 kDa, the number-average molecular weight of 694 kDa, and the polydispersity index of 2.11. Congo red experiment confirmed the absence of a triple helix conformation. Scanning electron microscopy showed that EPS53 displayed a three-dimensional fibrous structure covered with flakes. The in vitro findings indicated that EPS53 enhanced phagocytosis ability, reactive oxygen species (ROS) production, and cytokine levels of macrophages via the TLR4-mediated NF-κB/MAPK signaling pathways as confirmed by immunofluorescence staining experiments, inhibition blocking experiments, and Western blot assay. Additionally, the in vivo experiments demonstrated that EPS53 significantly increased macrophage and neutrophil number while enhancing NO and ROS levels in zebrafish larvae; thus, providing further evidence for the immunomodulatory efficacy of EPS53.

Extracellular vesicles from Candida albicans modulate immune cells function and play a protective role in fungal keratitis

Fungal keratitis (FK) is a highly blinding infectious corneal disease caused by pathogenic fungi. Candida albicans (C. albicans) is one of the main pathogens of fungal keratitis. Extracellular vesicles (EVs), lipid bilayer compartments released by almost all living cells, including fungi, have garnered attention for their role in pathogenic microbial infection and host immune responses in recent years. Studies have reported that pretreating the host with fungal EVs can reduce the inflammatory response of the host when attacked by fungi and reduce the lethality of fungal infection. However, there are no studies that have evaluated whether C. albicans EVs can modulate the inflammatory response associated with C. albicans keratitis. Our study revealed that C. albicans EVs could activate the polymorphonuclear cells (PMNs) and promote their secretion of proinflammatory cytokines and nitric oxide (NO), enhance their phagocytic and fungicidal abilities against C. albicans. C. albicans EVs also induced a proinflammatory response in RAW264.7 cells, which is characterized by increased production of inflammatory cytokines and elevated expression of the chemokine CCL2. Similarly, stimulation of C. albicans EVs to RAW264.7 cells also enhanced the phagocytosis and killing ability of cells against C. albicans. Besides, in vivo experiments, after receiving subconjunctival injection of C. albicans EVs, C57BL/6 mice were infected with C. albicans. The results demonstrated that pre-exposure to C. albicans EVs could effectively diminish the severity of keratitis, reduce fungal load and improve prognosis. Overall, we conclude that C. albicans EVs can modulate the function of immune cells and play a protective role in C. albicans keratitis.

TREM2 alleviates white matter injury after traumatic brain injury in mice might be mediated by regulation of DHCR24/LXR pathway in microglia.

White matter injury (WMI) is an important pathological process after traumatic brain injury (TBI). The correlation between white matter functions and the myeloid cells expressing triggering receptor-2 (TREM2) has been convincingly demonstrated. Moreover, a recent study revealed that microglial sterol metabolism is crucial for early remyelination after demyelinating diseases. However, the potential roles of TREM2 expression and microglial sterol metabolism in WMI after TBI have not yet been explored. Controlled cortical injury was induced in both wild-type (WT) and TREM2 depletion (TREM2 KO) mice to simulate clinical TBI. COG1410 was used to upregulate TREM2, while PLX5622 and GSK2033 were used to deplete microglia and inhibit the liver X receptor (LXR), respectively. Immunofluorescence, Luxol fast blue staining, magnetic resonance imaging, transmission electron microscopy, and oil red O staining were employed to assess WMI after TBI. Neurological behaviour tests and electrophysiological recordings were utilized to evaluate cognitive functions following TBI. Microglial cell sorting and transcriptomic sequencing were utilized to identify alterations in microglial sterol metabolism-related genes, while western blot was conducted to validate the findings. TREM2 expressed highest at 3 days post-TBI and was predominantly localized to microglial cells within the white matter. Depletion of TREM2 worsened aberrant neurological behaviours, and this phenomenon was mediated by the exacerbation of WMI, reduced renewal of oligodendrocytes, and impaired phagocytosis ability of microglia after TBI. Subsequently, the upregulation of TREM2 alleviated WMI, promoted oligodendrocyte regeneration, and ultimately facilitated the recovery of neurological behaviours after TBI. Finally, the expression of DHCR24 increased in TREM2 KO mice after TBI. Interestingly, TREM2 inhibited DHCR24 and upregulated members of the LXR pathway. Moreover, LXR inhibition could partially reverse the effects of TREM2 upregulation on electrophysiological activities. We demonstrate that TREM2 has the potential to alleviate WMI following TBI, possibly through the DHCR24/LXR pathway in microglia.

Decreased neutrophil function in newly diagnosed multiple myeloma patients is restored with lenalidomide therapy.

Bacterial infections are common and a major cause of morbidity and mortality in multiple myeloma (MM). We have investigated the function of polymorphonuclear leukocyte (PMN), the immune system's first line of defense against bacteria, in peripheral blood (PB) and bone marrow (BM) samples from patients with newly diagnosed MM (NDMM), smoldering MM (SMM), monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Phagocytosis and oxidative burst in PMN cells from patients and healthy donors were investigated using PhagoTest and PhagoBurst assay. PMN from NDMM, SMM, and MGUS patients had reduced phagocytosis and oxidative burst ability compared with healthy controls. The dysfunction was most prominent in BM samples from MM, SMM, and MGUS patients. Importantly the reduced phagocytosis in MM patients was restored in patients on lenalidomide therapy. Consistently the ability of Escherichia coli stimulated oxidative burst in BM was reduced for the MM, SMM, and MGUS cohort in contrast to the healthy controls and the patients on lenalidomide treatment. Our results show that MM patients have neutrophil dysfunction that could contribute to susceptibility for bacterial infections and that lenalidomide therapy was associated with restored PMN function.

A non-immunotoxic exopolysaccharide with anticancer effect in colorectal cancer isolated from Lacticaseibacillus rhamnosus ACS5 strain

Recent studies demonstrated the potential of exopolysaccharides (EPS) produced by LAB (lactic acid bacteria), to be used in different fields. In our study, L-EPS-ACS5 from Lacticaseibacillus rhamnosus ACS5 strain isolated from Ayvalık Cunda Sepet Cheese, one of the traditional cheeses of Turkey, showed antiproliferative effect in a time-dependent manner in human colon cancer (HT-29) cell line compared to healthy control line (L929). This effect was also confirmed by colony formation analysis. Apoptotic changes were investigated at molecular level using qPCR, Western blot and it was determined that the relevant L-EPS induced apoptosis by increasing Bax, Caspase 3, and 9 expression while reducing the Bcl-2 expression. These changes were confirmed by observations done at nuclear and cellular morphology level using a fluorescence microscope (Hoescht 33258) and TEM. In addition, the immunostimulatory activity of the relevant L-EPS was evaluated in terms of proliferation, phagocytosis ability and nitric oxide (NO) production parameters in the RAW 264.7 macrophage cell line. The increase in proliferative activity was not significant, and in parallel with these results, the differences in the control and application groups as a result of 'phagocytic activity and NO production analyzes' were also not significant. However, L-EPS-ACS5 did not show immunotoxic/cytotoxic activity in macrophage cells (% viability >70). All these cellular and molecular evidences indicate that L-EPS-ACS5 may be a good alternative to synthetic anti-cancer agents with its anticancer effect and non-immunotoxic properties in colorectal cancer.

Immunoenhancement effect of cinobufagin on macrophages and the cyclophosphamide-induced immunosuppression mouse model

Cinobufagin (CBG) is a natural active substance. Although its various pharmacological activities have been explored, the immunomodulatory activity of CBG remains unexplored. Therefore, this study aimed to investigate the anti-inflammatory and immunomodulatory activities of CBG ex vivo and in vivo. The immunomodulatory activity of CBG was investigated in RAW 264.7 cells. CBG showed no significant toxicity to cells. Additionally, 0.5–8 μg/mL CBG significantly increased the phagocytosis ability of macrophages and the secretion levels of IL-1β and TNF-α. Thus, it exerted immunomodulatory effects. We established the immunosuppressive model induced by cyclophosphamide (CTX) in mice and studied the immunomodulatory activity of CBG in vivo. The experimental results showed that the intervention of CBG alleviated the CTX-induced weight loss, restored the lymphocyte nuclear cell number, and promoted the secretion and mRNA expression of cytokines IFN-γ, IL-4, IL-6, and IL-12. Moreover, CBG increased the immune organ index, protected the growth of the spleen and thymus, and improved the pathological changes in immunosuppressed mice. Western blot results showed that different concentrations of CBG upregulated the phosphorylation level of PI3K/Akt/mTOR in the spleen of CTX-induced immunosuppressed mice. This suggests that the immunomodulatory effect of CBG may be related to the regulation of PI3K/Akt/mTOR signaling pathway. This study provides a theoretical basis for developing CBG immune enhancers and opens up new ideas for the comprehensive utilization and development of CBG in factories.

Targeting aberrant glycosylation to modulate microglial response and improve cognition in models of Alzheimer’s disease

Altered glycosylation profiles have been correlated with potential drug targets in various diseases, including Alzheimer’s disease (AD). In this area, the linkage between bisecting N-acetylglucosamine (GlcNAc), a product of N-acetylglucosaminyltransferase III (GnT-III), and AD has been recognized, however, our understanding of the cause and the causative role of this aberrant glycosylation in AD are far from completion. Moreover, the effects and mechanisms of glycosylation-targeting interventions on memory and cognition, and novel targeting strategies are worth further study. Here, we showed the characteristic amyloid pathology-induced and age-related changes of GnT-III, and identified transcription factor 7-like 2 as the key transcription factor responsible for the abnormal expression of GnT-III in AD. Upregulation of GnT-III aggravated cognitive dysfunction and Alzheimer-like pathologies. In contrast, loss of GnT-III could improve cognition and alleviate pathologies. Furthermore, we found that an increase in bisecting GlcNAc modified ICAM-1 resulted in impairment of microglial responses, and genetic inactivation of GnT-III protected against AD mechanistically by blocking the aberrant glycosylation of ICAM-1 and subsequently modulating microglial responses, including microglial motility, phagocytosis ability, homeostatic/reactive state and neuroinflammation. Moreover, by target-based screening of GnT-III inhibitors from FDA-approved drug library, we identified two compounds, regorafenib and dihydroergocristine mesylate, showing pharmacological potential leading to modulation of aberrant glycosylation and microglial responses, and rescue of memory and cognition deficits.

Mechanism of Chinese sturgeon IFN-γ inhibition on Mycobacterium marinum (Acipenser sinensis)

IFN-γ plays a crucial role in both innate and adaptive immune responses and is a typical Th1 cytokine that promotes Th1 response and activates macrophages. When macrophages were incubated with IFN-γ, their phagocytosis ratio against Mycobacterium marinum increased significantly, as observed under fluorescence microscopy. The macrophages engulfed a large number of M. marinum. The proliferative ability of macrophages treated with IFN-γ was significantly weaker on the 4th and 7th day after phagocytosis and subsequent re-infection with marine chlamydia (P < 0.001). This suggests that IFN-γ enhances the phagocytosis and killing ability of macrophages against M. marinum. IFN-γ protein also significantly increased the production of reactive oxygen species (H2O2) and nitric oxide (NO) by macrophages. Additionally, the expression levels of toll-like receptor 2 (tlr2) and caspase 8 (casp8) were significantly higher in macrophages after IFN-γ incubation compared to direct infection after 12 h of M. marinum stimulation. Apoptosis was also observed to a higher degree in IFN-γ incubated macrophage. Moreover, mRNA expression of major histocompatibility complex (MHC) molecules produced by macrophages after IFN-γ incubation was significantly higher than direct infection. This indicates that IFN-γ enhances antigen presentation by upregulating MHC expression. It also upregulates tlr2 and casp8 expression through the TLR2 signaling pathway to induce apoptosis in macrophages. The pro-inflammatory cytokine showed an initial increase followed by a decline, suggesting that IFN-γ enhances the immune response of macrophages against M. marinum infection. On the other hand, the anti-inflammatory cytokine showed a delayed increase, significantly reducing the expression of pro-inflammatory cytokines. The expression of both cytokines balanced each other and together regulated the inflammatory reaction against M. marinum infection.

Neutrophil-like cells derived from the HL-60 cell-line as a genetically-tractable model for neutrophil degranulation.

Research on neutrophil biology has been limited by the short life span and limited genetic manipulability of these cells, driving the need for representative and efficient model cell lines. The promyelocytic cell line HL-60 and its subline PLB-985 can be differentiated into neutrophil-like cells (NLCs) and have been used to study neutrophil functions including chemotaxis, phagocytosis, endocytosis, and degranulation. Compared to neutrophils derived from hematopoietic stem cells, NLCs serve as a cost-effective neutrophil model. NLCs derived from both HL-60 and PLB-985 cells have been shown to perform degranulation, an important neutrophil function. However, no study has directly compared the two lines as models for degranulation including their release of different types of mobilizable organelles. Furthermore, Nutridoma, a commercially available supplement, has recently been shown to improve the chemotaxis, phagocytosis, and oxidative burst abilities of NLCs derived from promyelocytic cells, however it is unknown whether this reagent also improves the degranulation ability of NLCs. Here, we show that NLCs derived from both HL-60 and PLB-985 cells are capable of degranulating, with each showing markers for the release of multiple types of secretory organelles, including primary granules. We also show that differentiating HL-60 cells using Nutridoma does not enhance their degranulation activity over NLCs differentiated using Dimethyl Sulfoxide (DMSO) plus Granulocyte-colony stimulating factor (G-CSF). Finally, we show that promyelocytic cells can be genetically engineered and differentiated using these methods, to yield NLCs with a defect in degranulation. Our results indicate that both cell lines serve as effective models for investigating the mechanisms of neutrophil degranulation, which can advance our understanding of the roles of neutrophils in inflammation and immunity.

Cell fusion between tumor cells and macrophages promotes the metastasis of OSCC patient through the activation of the chemokine signaling pathway.

Tumor metastasis is responsible for the high mortality rate of patients with oral squamous cell carcinoma (OSCC). Although many hypotheses have been proposed to elucidate the mechanism of tumor metastasis, the origin of the metastatic tumor cells remains unclear. In this study, we explored the role of cell fusion in the formation of OSCC metastatic tumor cells. Murine OSCC tumor cells and macrophages were fused invitro, and the cell proliferation, migration, and phagocytosis abilities of hybrid cells and parental cells were compared. Subsequently, we compared the transcriptome differences between hybrid and parental cells. Murine OSCC tumor cells and macrophages were successfully fused invitro. The cytological and molecular experimental results revealed that OSCC tumor cells obtained a migration-related phenotype after fusion with macrophages, and the migration ability of hybrid cells was related to the activation of the "chemokine signal pathway". After fusion with macrophages, the chemokine signaling pathway in OSCC tumor cells was activated, leading to metastasis.