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Frontiers in Immunology 2024Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal...
INTRODUCTION
Protein kinases are indispensable reversible molecular switches that adapt and control protein functions during cellular processes requiring rapid responses to internal and external events. Bacterial infections can affect kinase-mediated phosphorylation events, with consequences for both innate and adaptive immunity, through regulation of antigen presentation, pathogen recognition, cell invasiveness and phagocytosis. (), a human respiratory tract pathogen and a major cause of community-acquired pneumoniae, affects phosphorylation-based signalling of several kinases, but the pneumococcal mediator(s) involved in this process remain elusive. In this study, we investigated the influence of pneumococcal HO on the protein kinase activity of the human lung epithelial H441 cell line, a generally accepted model of alveolar epithelial cells.
METHODS
We performed kinome analysis using PamGene microarray chips and protein analysis in Western blotting in H441 lung cells infected with wild type () or with -a deletion mutant strongly attenuated in HO production- to assess the impact of pneumococcal hydrogen peroxide (HO) on global protein kinase activity profiles.
RESULTS
Our kinome analysis provides direct evidence that kinase activity profiles in infected H441 cells significantly vary according to the levels of pneumococcal HO. A large number of kinases in H441 cells infected with are significantly downregulated, whereas this no longer occurs in cells infected with the mutant strain, which lacks HO In particular, we describe for the first time HO-mediated downregulation of Protein kinase B (Akt1) and activation of lymphocyte-specific tyrosine protein kinase (Lck) via HO-mediated phosphorylation.
Topics: Streptococcus pneumoniae; Hydrogen Peroxide; Humans; Phosphorylation; Host-Pathogen Interactions; Cell Line; Protein Kinases; Pneumococcal Infections; Signal Transduction
PubMed: 38903521
DOI: 10.3389/fimmu.2024.1414195 -
Frontiers in Immunology 2024Human milk oligosaccharides (HMOs) are present in high numbers in milk of lactating women. They are beneficial to gut health and the habitant microbiota, but less is...
Human milk oligosaccharides (HMOs) are present in high numbers in milk of lactating women. They are beneficial to gut health and the habitant microbiota, but less is known about their effect on cells from the immune system. In this study, we investigated the direct effect of three structurally different HMOs on human derived macrophages before challenge with (). The study demonstrates that individual HMO structures potently affect the activation, differentiation and development of monocyte-derived macrophages in response to . 6´-Sialyllactose (6'SL) had the most pronounced effect on the immune response against , as illustrated by altered expression of macrophage surface markers, pointing towards an activated M1-like macrophage-phenotype. Similarly, 6'SL increased production of the pro-inflammatory cytokines TNF-α, IL-6, IL-8, IFN-γ and IL-1β, when exposing cells to 6'SL in combination with compared with alone. Interestingly, macrophages treated with 6'SL exhibited an altered proliferation profile and increased the production of the classic M1 transcription factor NF-κB. The HMOs also enhanced macrophage phagocytosis and uptake of . Importantly, the different HMOs did not notably affect macrophage activation and differentiation without exposure. Together, these findings show that HMOs can potently augment the immune response against , without causing inflammatory activation in the absence of , suggesting that HMOs assist the immune system in targeting important pathogens during early infancy.
Topics: Humans; Milk, Human; Staphylococcus aureus; Macrophages; Oligosaccharides; Macrophage Activation; Cytokines; Phagocytosis; Female; Cell Differentiation; Staphylococcal Infections; Cells, Cultured
PubMed: 38903508
DOI: 10.3389/fimmu.2024.1379042 -
Frontiers in Immunology 2024Extracellular vesicles (EVs), characterized by low immunogenicity, high biocompatibility and targeting specificity along with excellent blood-brain barrier permeability,... (Review)
Review
Extracellular vesicles (EVs), characterized by low immunogenicity, high biocompatibility and targeting specificity along with excellent blood-brain barrier permeability, are increasingly recognized as promising drug delivery vehicles for treating a variety of diseases, such as cancer, inflammation and viral infection. However, recent findings demonstrate that the intracellular delivery efficiency of EVs fall short of expectations due to phagocytic clearance mediated by the host mononuclear phagocyte system through Fcγ receptors, complement receptors as well as non-opsonic phagocytic receptors. In this text, we investigate a range of bacterial virulence proteins that antagonize host phagocytic machinery, aiming to explore their potential in engineering EVs to counteract phagocytosis. Special emphasis is placed on IdeS secreted by and ImpA secreted by , as they not only counteract phagocytosis but also bind to highly upregulated surface biomarkers αβ on cancer cells or cleave the tumor growth and metastasis-promoting factor CD44, respectively. This suggests that bacterial anti-phagocytic proteins, after decorated onto EVs using pre-loading or post-loading strategies, can not only improve EV-based drug delivery efficiency by evading host phagocytosis and thus achieve better therapeutic outcomes but also further enable an innovative synergistic EV-based cancer therapy approach by integrating both phagocytosis antagonism and cancer targeting or deactivation.
Topics: Extracellular Vesicles; Phagocytosis; Humans; Animals; Bacterial Proteins; Neoplasms; Integrin alphaVbeta3; Hyaluronan Receptors; Pseudomonas aeruginosa
PubMed: 38903499
DOI: 10.3389/fimmu.2024.1418061 -
Clinical Endoscopy Jun 2024During endoscopy, white spots (WS) are sometimes observed around benign or malignant colorectal tumors; however, few reports have investigated WS, and their significance...
BACKGROUND/AIMS
During endoscopy, white spots (WS) are sometimes observed around benign or malignant colorectal tumors; however, few reports have investigated WS, and their significance remains unknown. Therefore, we investigated the significance of WS from clinical and pathological viewpoints and evaluated its usefulness in endoscopic diagnosis.
METHODS
Clinical data of patients with lesions diagnosed as epithelial tumors from January 1, 2019, to December 31, 2020, were analyzed (n=3,869). We also performed a clinicopathological analysis of adenomas or carcinomas treated with endoscopic resection (n=759). Subsequently, detailed pathological observations of the WS were performed.
RESULTS
The positivity rates for WS were 9.3% (3,869 lesions including advanced cancer and non-adenoma/carcinoma) and 25% (759 lesions limited to adenoma and early carcinoma). Analysis of 759 lesions showed that the WS-positive lesion group had a higher proportion of cancer cases and larger tumor diameters than the WS-negative group. Multiple logistic analysis revealed the following three statistically significant risk factors for carcinogenesis: positive WS, flat lesions, and tumor diameter ≥5 mm. Pathological analysis revealed that WS were macrophages that phagocytosed fat and mucus and were white primarily because of fat.
CONCLUSIONS
WS are cancer-related findings and can become a new criterion for endoscopic resection in the future.
PubMed: 38902852
DOI: 10.5946/ce.2024.027 -
Frontiers in Immunology 2024is a common Gram-negative bacterium. Blood infection caused by is one of the most common causes of human sepsis, which seriously threatens the life of patients. The...
BACKGROUND
is a common Gram-negative bacterium. Blood infection caused by is one of the most common causes of human sepsis, which seriously threatens the life of patients. The immune status of peripheral blood mononuclear cells (PBMCs) based on single-cell RNA sequencing (scRNA-seq) in acute stage and recovery stage of sepsis caused by bloodstream infection has not been studied.
METHODS
A total of 13 subjects were included in this study, 3 healthy controls, 7 patients with bloodstream infection in the acute stage (4 patients died), and 3 patients in the recovery stage. Peripheral blood of all patients was collected and PBMCs were isolated for scRNA-seq analysis. We studied the changes of PBMCs components, signaling pathways, differential genes, and cytokines in acute and recovery stages.
RESULTS
During acute infection we observed a decrease in the proportion of T cells, most probably due to apoptosis and the function of T cell subtypes was disorder. The proportion of monocytes increased in acute stage. Although genes related to their phagocytosis function were upregulated, their antigen presentation capacity-associated genes were downregulated. The expression of IL-1β, IL-18, IFNGR1 and IFNGR2 genes was also increased in monocytes. The proportion of DCs was depleted during the acute stage and did not recover during sepsis recovery. DCs antigen presentation was weakened during the acute stage but recovered fast during the recovery stage. pDCs response to MCP-1 chemokine was weakened, they recovered it quickly during the recovery stage. B cells showed apoptosis both in the acute stage and recovery stage. Their response to complement was weakened, but their antigen presentation function was enhanced. The proportion of NK cells stable during all disease's stages, and the expression of IFN-γ gene was upregulated.
CONCLUSION
The proportion of PBMCs and their immune functions undergo variations throughout the course of the disease, spanning from the acute stage to recovery. These findings provide new insights into the mechanism of PBMCs immune function during bloodstream infection sepsis and recovery and sets the basis for further understanding and treatment.
Topics: Humans; Klebsiella pneumoniae; Klebsiella Infections; Leukocytes, Mononuclear; Male; Female; Middle Aged; Sepsis; Aged; Single-Cell Analysis; Cytokines; Bacteremia; Sequence Analysis, RNA; Adult
PubMed: 38898888
DOI: 10.3389/fimmu.2024.1380211 -
Cell Communication and Signaling : CCS Jun 2024Excessive scar formation such as hypertrophic scars and keloids, resulting from trauma or surgical procedures, present a widespread concern for causing disfigurement,...
Excessive scar formation such as hypertrophic scars and keloids, resulting from trauma or surgical procedures, present a widespread concern for causing disfigurement, discomfort, and functional limitations. Macrophages play pivotal roles in maintaining tissue homeostasis, orchestrating tissue development, repair, and immune responses, and its transition of function and phenotype plays a critical role in regulating the balance between inflammation and tissue regeneration, which is central to cutaneous scar formation. Recent evidence suggests the involvement of Sonic Hedgehog (SHH) in the induction of anti-inflammatory M2-like macrophage phenotypes within tumor microenvironments. In our study, we observed increased SHH expression in human hypertrophic scars, prompting an investigation into its influence on macrophage polarization, efferocytosis, and cutaneous scar formation. Our findings reveal that SHH can enhance oxidative phosphorylation (OXPHOS) in macrophages, augment macrophage efferocytosis, and promote M2 polarization, finally contributing to the progression of cutaneous scar formation. Notably, targeting SHH signaling with vismodegib exhibited promising potential in mitigating scar formation by reversing the effects of enhanced OXPHOS and M2 polarization in macrophages. In conclusion, this study underscores the critical roles of macrophage metabolism, particularly OXPHOS, efferocytosis and SHH signaling in cutaneous scar formation. Understanding these mechanisms provides new avenues for potential interventions and scar prevention strategies.
Topics: Hedgehog Proteins; Macrophages; Humans; Oxidative Phosphorylation; Animals; Phagocytosis; Cicatrix, Hypertrophic; Mice; Signal Transduction; Cicatrix; Mice, Inbred C57BL; Anilides; Pyridines; Efferocytosis
PubMed: 38898530
DOI: 10.1186/s12964-024-01692-w -
BioRxiv : the Preprint Server For... Jun 2024CLN3 Batten disease (also known as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but...
PURPOSE
CLN3 Batten disease (also known as Juvenile Neuronal Ceroid Lipofuscinosis; JNCL) is a lysosomal storage disorder that typically initiates with retinal degeneration but is followed by seizure onset, motor decline and premature death. Patient-derived CLN3 disease iPSC-RPE cells show defective phagocytosis of photoreceptor outer segments (POSs). Because modifier genes are implicated in CLN3 disease, our goal here was to investigate a direct link between mutation and POS phagocytosis defect.
METHODS
Isogenic control and mutant stem cell lines were generated by CRISPR-Cas9-mediated biallelic deletion of exons 7 and 8. A transgenic ( ) Yucatan miniswine was also used to study the impact of mutation on POS phagocytosis. POS phagocytosis by cultured RPE cells was analyzed by Western blotting and immunohistochemistry. Electroretinogram, optical coherence tomography and histological analysis of and wild-type miniswine eyes were carried out at 6-, 36-, or 48-month age.
RESULTS
RPE ( RPE) displayed reduced POS binding and consequently decreased uptake of POS compared to isogenic control RPE cells. Furthermore, wild-type miniswine RPE cells phagocytosed POS less efficiently than wild-type POS. Consistent with decreased POS phagocytosis, lipofuscin/autofluorescence was decreased in miniswine RPE at 36 months-of-age and was followed by almost complete loss of photoreceptors at 48 months of age.
CONCLUSIONS
mutation (that affects up to 85% patients) affects both RPE and POSs and leads to photoreceptor cell loss in CLN3 disease. Furthermore, both primary RPE dysfunction and mutant POS independently contribute to impaired POS phagocytosis in CLN3 disease.
PubMed: 38895469
DOI: 10.1101/2024.06.09.597388 -
BioRxiv : the Preprint Server For... Jun 2024Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors....
UNLABELLED
Chimeric antigen receptor (CAR) T-cell therapy has revolutionized the treatment of hematological malignancies but has been clinically less effective in solid tumors. Engineering macrophages with CARs has emerged as a promising approach to overcome some of the challenges faced by CAR-T cells due to the macrophage's ability to easily infiltrate tumors, phagocytose their targets, and reprogram the immune response. We engineered CAR-macrophages (CAR-Ms) to target chondroitin sulfate proteoglycan 4 (CSPG4), an antigen expressed in melanoma, and several other solid tumors. CSPG4-targeting CAR-Ms exhibited specific phagocytosis of CSPG4-expressing melanoma cells. Combining CSPG4-targeting CAR-Ms with CD47 blocking antibodies synergistically enhanced CAR-M-mediated phagocytosis and effectively inhibited melanoma spheroid growth in 3D. Furthermore, CSPG4-targeting CAR-Ms inhibited melanoma tumor growth in mouse models. These results suggest that CSPG4-targeting CAR-M immunotherapy is a promising solid tumor immunotherapy approach for treating melanoma.
STATEMENT OF SIGNIFICANCE
We engineered macrophages with CARs as an alternative approach for solid tumor treatment. CAR-macrophages (CAR-Ms) targeting CSPG4, an antigen expressed in melanoma and other solid tumors, phagocytosed melanoma cells and inhibited melanoma growth . Thus, CSPG4-targeting CAR-Ms may be a promising strategy to treat patients with CSPG4-expressing tumors.
PubMed: 38895447
DOI: 10.1101/2024.06.04.597413 -
Cancers May 2024Recent studies highlight the integral role of the interferon gamma receptor (IFNγR) pathway in T cell-mediated cytotoxicity against solid but not liquid tumors. IFNγ...
Recent studies highlight the integral role of the interferon gamma receptor (IFNγR) pathway in T cell-mediated cytotoxicity against solid but not liquid tumors. IFNγ not only directly facilitates tumor cell death by T cells but also indirectly promotes cytotoxicity via myeloid phagocytosis in the tumor microenvironment. Meanwhile, full human ex vivo immune checkpoint drug screening remains challenging. We hypothesized that an engineered gamma interferon activation site response element luciferase reporter (GAS-Luc2) can be utilized for immune checkpoint drug screening in diverse ex vivo T cell-solid tumor cell co-culture systems. We comprehensively profiled cell surface proteins in ATCC's extensive collection of human tumor and immune cell lines, identifying those with endogenously high expression of established and novel immune checkpoint molecules and binding ligands. We then engineered three GAS-Luc2 reporter tumor cell lines expressing immune checkpoints PD-L1, CD155, or B7-H3/CD276. Luciferase expression was suppressed upon relevant immune checkpoint-ligand engagement. In the presence of an immune checkpoint inhibitor, T cells released IFNγ, activating the JAK-STAT pathway in GAS-Luc2 cells, and generating a quantifiable bioluminescent signal for inhibitor evaluation. These reporter lines also detected paracrine IFNγ signaling for immune checkpoint-targeted ADCC drug screening. Further development into an artificial antigen-presenting cell line (aAPC) significantly enhanced T cell signaling for superior performance in these ex vivo immune checkpoint drug screening platforms.
PubMed: 38893085
DOI: 10.3390/cancers16111965 -
Nutrients May 2024Longan ( Lour.) is a kind of traditional fruit used as a medicine and a food. Fresh longan is primarily consumed as a fruit, whereas dried longan is commonly employed... (Comparative Study)
Comparative Study
Longan ( Lour.) is a kind of traditional fruit used as a medicine and a food. Fresh longan is primarily consumed as a fruit, whereas dried longan is commonly employed for medicinal purposes. The differences in the immunomodulatory activities and mechanisms of polysaccharides between dried and fresh longan remain unclear. The present study comparatively analyzed the mechanisms of macrophage activation induced by polysaccharides from dried (LPG) and fresh longan (LPX). The results revealed that LPG and LPX differentially promoted macrophage phagocytosis and the secretion of NO, TNF-α, and IL-6. RNA-seq analysis revealed that LPG and LPX differentially affected gene expression in macrophages. The LPG treatment identified and chemokine-related genes as core genes, while and interferon-related genes were the core genes affected by LPX. A comprehensive analysis of the differentially expressed genes showed that LPG initiated macrophage activation primarily through the TLR2/4-mediated and CLR-mediated NF-κB signaling pathways. LPX initiated macrophage activation predominantly via the CLR-mediated and NLR-mediated MAPK and NF-κB signaling pathways. Interestingly, the non-classical NF-κB signaling pathway was activated by polysaccharides in both dried and fresh longan to elicit a slow, mild immune response. LPG tends to promote immune cell migration to engage in the immune response, while LPX facilitates antigen presentation to promote T cell activation. These findings contribute insights into the mechanisms underlying the differences in bioactivity between dried and fresh longan and their potential applications in immune-enhancing strategies and functional-food development.
Topics: Macrophage Activation; Polysaccharides; Animals; Mice; RAW 264.7 Cells; Macrophages; Signal Transduction; Fruit; Sapindaceae; Phagocytosis; NF-kappa B
PubMed: 38892587
DOI: 10.3390/nu16111654