Similarities and differences between brain and skin GNAQ p.R183Q driven capillary malformations.

Capillary malformations (CM) are congenital vascular irregularities of capillary and venous blood vessels that appear in the skin, leptomeninges of the brain, and the choroid of the eye in the disorder known as Sturge Weber Syndrome (SWS). More common are non-syndromic CM found only in the skin, without brain or ocular involvement. A somatic activating mutation in GNAQ (p.R183Q) is found in ~90% of syndromic and non-syndromic CM specimens and is present in CD31pos endothelial cells isolated from brain and skin CM specimens. Endothelial expression of the GNAQ p.R183Q variant is sufficient to form CM-like vessels in mice. Given the distinct features and functions of blood vessels in the brain versus the skin, we examined the features of CM vessels in both tissues to gain insights into the pathogenesis of CM. Herein, we present morphologic characteristics of CM observed in specimen from brain and skin. The GNAQ p.R183Q variant allelic frequency in each specimen was determined by droplet digital PCR. Sections were stained for endothelial cells, tight junctions, mural cells, and macrophages to assess the endothelium as well as perivascular constituents. CM blood vessels in brain and skin were enlarged, exhibited fibrin leakage and reduced zona occludin-1, and were surrounded by MRC1pos/LYVE1pos macrophages. In contrast, the CMs from brain and skin differ in endothelial sprouting activity and localization of mural cells. These characteristics might be helpful in the development of targeted and/or tissue specific therapies to prevent or reverse non-syndromic and syndromic CM.

Tumor-associated macrophages in anti-PD-1/PD-L1 immunotherapy for hepatocellular carcinoma: recent research progress.

Hepatocellular carcinoma (HCC) is one of the cancers that seriously threaten human health. Immunotherapy serves as the mainstay of treatment for HCC patients by targeting the programmed cell death protein 1/programmed cell death 1 ligand 1 (PD-1/PD-L1) axis. However, the effectiveness of anti-PD-1/PD-L1 treatment is limited when HCC becomes drug-resistant. Tumor-associated macrophages (TAMs) are an important factor in the negative regulation of PD-1 antibody targeted therapy in the tumor microenvironment (TME). Therefore, as an emerging direction in cancer immunotherapy research for the treatment of HCC, it is crucial to elucidate the correlations and mechanisms between TAMs and PD-1/PD-L1-mediated immune tolerance. This paper summarizes the effects of TAMs on the pathogenesis and progression of HCC and their impact on HCC anti-PD-1/PD-L1 immunotherapy, and further explores current potential therapeutic strategies that target TAMs in HCC, including eliminating TAMs in the TME, inhibiting TAMs recruitment to tumors and functionally repolarizing M2-TAMs (tumor-supportive) to M1-TAMs (antitumor type).

Critical role of model organism selection in assessing weak urban electromagnetic field effects: Implications for human health.

The impact of electromagnetic fields on human health has been investigated in recent years using various model organisms, yet the findings remain unclear. In our work, we examined the effect of less-explored, weak electromagnetic fields commonly found in the urban environments we inhabit. We studied different impacts of electromagnetic fields with a frequency of 50 Hz and a combination of 50 Hz and 150 Hz, on both yeasts (Saccharomyces cerevisiae) and human macrophages. We determined growth, survival, and protein composition (SDS-PAGE) (Saccharomyces cerevisiae) and morphology of macrophages (human monocytic cell line). In yeast, the sole observed change after 24 h of exposure was the extension of the exponential growth phase by 17 h. Conversely, macrophages exhibited morphological transformations from the anti-inflammatory to the pro-inflammatory type within just 2 h of exposure to the electromagnetic field. Our results suggest that effects of electromagnetic field largely depend on the model organism. The selection of an appropriate model organism proves essential for the study of the specific impacts of electromagnetic fields. The potential risk associated with the presence of pro-inflammatory M1 macrophages in everyday urban environments primarily arises from the continual promotion of inflammatory reactions within a healthy organism and deserves further investigation.

Hypoxia and inflammation induce synergistic transcriptome turnover in macrophages.

Macrophages are effector immune cells that experience substantial changes to oxygenation when transiting through tissues, especially when entering tumors or infected wounds. How hypoxia alters gene expression and macrophage effector function at the post-transcriptional level remains poorly understood. Here, we use TimeLapse-seq to measure how inflammatory activation modifies the hypoxic response in primary macrophages. Nucleoside recoding sequencing allows the derivation of steady-state transcript levels, degradation rates, and transcriptional synthesis rates from the same dataset. We find that hypoxia produces distinct responses from resting and inflammatory macrophages. Hypoxia induces destabilization of mRNA transcripts, though inflammatory macrophages substantially increase mRNA degradation compared to resting macrophages. Increased RNA turnover results in the upregulation of ribosomal protein genes and downregulation of extracellular matrix components in inflammatory macrophages. Pathways regulated by mRNA decay in vitro are differentially regulated in tumor-associated macrophages implying that mixed stimuli could induce post-transcriptional regulation of macrophage function in solid tumors.

The role of macrophages in fibrosis of chronic kidney disease.

Macrophages are widely distributed throughout various tissues of the body, and mounting evidence suggests their involvement in regulating the tissue microenvironment, thereby influencing disease onset and progression through direct or indirect actions. In chronic kidney disease (CKD), disturbances in renal functional homeostasis lead to inflammatory cell infiltration, tubular expansion, glomerular atrophy, and subsequent renal fibrosis. Macrophages play a pivotal role in this pathological process. Therefore, understanding their role is imperative for investigating CKD progression, mitigating its advancement, and offering novel research perspectives for fibrosis treatment from an immunological standpoint. This review primarily delves into the intrinsic characteristics of macrophages, their origins, diverse subtypes, and their associations with renal fibrosis. Particular emphasis is placed on the transition between M1 and M2 phenotypes. In late-stage CKD, there is a shift from the M1 to the M2 phenotype, accompanied by an increased prevalence of M2 macrophages. This transition is governed by the activation of the TGF-ß1/SMAD3 and JAK/STAT pathways, which facilitate macrophage-to-myofibroblast transition (MMT). The tyrosine kinase Src is involved in both signaling cascades. By thoroughly elucidating macrophage functions and comprehending the modes and molecular mechanisms of macrophage-fibroblast interaction in the kidney, novel, tailored therapeutic strategies for preventing or attenuating the progression of CKD can be developed.

The significance of lipid metabolism reprogramming of tumor-associated macrophages in hepatocellular carcinoma.

In the intricate landscape of the tumor microenvironment, tumor-associated macrophages (TAMs) emerge as a ubiquitous cellular component that profoundly affects the oncogenic process. The microenvironment of hepatocellular carcinoma (HCC) is characterized by a pronounced infiltration of TAMs, underscoring their pivotal role in modulating the trajectory of the disease. Amidst the evolving therapeutic paradigms for HCC, the strategic reprogramming of metabolic pathways presents a promising avenue for intervention, garnering escalating interest within the scientific community. Previous investigations have predominantly focused on elucidating the mechanisms of metabolic reprogramming in cancer cells without paying sufficient attention to understanding how TAM metabolic reprogramming, particularly lipid metabolism, affects the progression of HCC. In this review article, we intend to elucidate how TAMs exert their regulatory effects via diverse pathways such as E2F1-E2F2-CPT2, LKB1-AMPK, and mTORC1-SREBP, and discuss correlations of TAMs with these processes and the characteristics of relevant pathways in HCC progression by consolidating various studies on TAM lipid uptake, storage, synthesis, and catabolism. It is our hope that our summary could delineate the impact of specific mechanisms underlying TAM lipid metabolic reprogramming on HCC progression and provide useful information for future research on HCC and the development of new treatment strategies.

Redefining the ontogeny of hyalocytes as yolk sac-derived tissue-resident macrophages of the vitreous body.

BACKGROUND: The eye is a highly specialized sensory organ which encompasses the retina as a part of the central nervous system, but also non-neural compartments such as the transparent vitreous body ensuring stability of the eye globe and a clear optical axis. Hyalocytes are the tissue-resident macrophages of the vitreous body and are considered to play pivotal roles in health and diseases of the vitreoretinal interface, such as proliferative vitreoretinopathy or diabetic retinopathy. However, in contrast to other ocular macrophages, their embryonic origin as well as the extent to which these myeloid cells might be replenished by circulating monocytes remains elusive. RESULTS: In this study, we combine transgenic reporter mice, embryonic and adult fate mapping approaches as well as parabiosis experiments with multicolor immunofluorescence labeling and confocal laser-scanning microscopy to comprehensively characterize the murine hyalocyte population throughout development and in adulthood. We found that murine hyalocytes express numerous well-known myeloid cell markers, but concomitantly display a distinct immunophenotype that sets them apart from retinal microglia. Embryonic pulse labeling revealed a yolk sac-derived origin of murine hyalocytes, whose precursors seed the developing eye prenatally. Finally, postnatal labeling and parabiosis established the longevity of hyalocytes which rely on Colony Stimulating Factor 1 Receptor (CSF1R) signaling for their maintenance, independent of blood-derived monocytes. CONCLUSION: Our study identifies hyalocytes as long-living progeny of the yolk sac hematopoiesis and highlights their role as integral members of the innate immune system of the eye. As a consequence of their longevity, immunosenescence processes may culminate in hyalocyte dysfunction, thereby contributing to the development of vitreoretinal diseases. Therefore, myeloid cell-targeted therapies that convey their effects through the modification of hyalocyte properties may represent an interesting approach to alleviate the burden imposed by diseases of the vitreoretinal interface.

Excessive palmitic acid disturbs macrophage α-ketoglutarate/succinate metabolism and causes adipose tissue insulin resistance associated with gestational diabetes mellitus.

Abnormal polarization of adipose tissue macrophages (ATMs) results in low-grade systemic inflammation and insulin resistance (IR), potentially contributing to the development of diabetes. However, the underlying mechanisms that regulate the polarization of ATMs associated with gestational diabetes mellitus (GDM) remain unclear. Thus, we aimed to determine the effects of abnormal fatty acids on macrophage polarization and development of insulin resistance in GDM. Levels of fatty acids and inflammation were assessed in the serum samples and adipose tissues of patients with GDM. An in vitro cell model treated with palmitic acid was established, and the mechanisms of palmitic acid in regulating macrophage polarization was clarified. The effects of excessive palmitic acid on the regulation of histone methylations and IR were also explored in the high-fat diet induced GDM mice model. We found that pregnancies with GDM were associated with increased levels of serum fatty acids, and inflammation and IR in adipose tissues. Increased palmitic acid could induce mitochondrial dysfunction and excessive ROS levels in macrophages, leading to abnormal cytoplasmic and nuclear metabolism of succinate and α-ketoglutarate (αKG). Specifically, a decreased nuclear αKG/succinate ratio could attenuate the enrichment of H3K27me3 at the promoters of pro-inflammatory cytokines, such as IL-1ß, IL-6, and TNF-α, leading to cytokine secretion. Importantly, GDM mice treated with GSK-J4, an inhibitor of histone lysine demethylase, were protected from abnormal pro-inflammatory macrophage polarization and excessive production of pro-inflammatory cytokines. Our findings highlight the importance of the metabolism of αKG and succinate as transcriptional modulators in regulating the polarization of ATMs and the insulin sensitivity of adipose tissue, ensuring a normal pregnancy. This novel insight sheds new light on gestational fatty acid metabolism and epigenetic alterations associated with GDM.

PRPS2-mediated modulation of the antitumor immune response in lung cancer through CCL2-mediated tumor-associated macrophages and myeloid-derived suppressor cells.

BACKGROUND: Phosphoribosyl pyrophosphate synthetase 2 (PRPS2) is known as an oncogene in many types of cancers, including lung cancer. However, its role in regulating tumor-associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) remains unclear. Our study aimed to explore the involvement of PRPS2 in TAM and MDSC regulation. METHODS: Stable Lewis lung cancer (LLC) cell lines were established using a lentivirus system. These LLC lines were then used to establish tumor model in mice. The levels of target genes were determined using qPCR, western blotting, and ELISA assays. The percentage of different immune cell types was analyzed using fluorescence-activated cell sorting. The chemotaxis ability of TAM and MDSC was evaluated using an in vitro transwell chemotaxis assay. RESULTS: Notably, PRPS2 was found to regulate the chemotaxis of TAM and MDSC in tumor cells, as evidenced by the positive correlation of PRPS2 expression levels and abundance of TAM and MDSC populations. In addition, the expression of CCL2, mediated by PRPS2, was identified as a key factor in the chemotaxis of TAM and MDSC, as evidenced by a significant reduction in macrophages and MDSC numbers in the presence of the CCL2 antibody. Furthermore, in vivo experiments confirmed the involvement of PRPS2 in mediating CCL2 expression. PRPS2 was also found to regulate immune cell infiltration into tumors, whereas knockdown of CCL2 reversed the phenotype induced by PRPS2 overexpression. In tumor tissues from mice implanted with LLC-PRPS2-shCCL2 cells, a notable increase in CD4+ and CD8+ T cell percentages, alongside a marked decrease in TAMs, M-MDSC, and PMN-MDSC, was observed. CONCLUSION: Taken together, PRPS2 plays a crucial role in modulating the antitumor immune response by reprogramming CCL2-mediated TAM and MDSC.

ASFV infection induces macrophage necroptosis and releases proinflammatory cytokine by ZBP1-RIPK3-MLKL necrosome activation.

African swine fever (ASF) is an infectious disease characterized by hemorrhagic fever, which is highly pathogenic and causes severe mortality in domestic pigs. It is caused by the African swine fever virus (ASFV). ASFV is a large DNA virus and primarily infects porcine monocyte macrophages. The interaction between ASFV and host macrophages is the major reason for gross pathological lesions caused by ASFV. Necroptosis is an inflammatory programmed cell death and plays an important immune role during virus infection. However, whether and how ASFV induces macrophage necroptosis and the effect of necroptosis signaling on host immunity and ASFV infection remains unknown. This study uncovered that ASFV infection activates the necroptosis signaling in vivo and macrophage necroptosis in vitro. Further evidence showed that ASFV infection upregulates the expression of ZBP1 and RIPK3 to consist of the ZBP1-RIPK3-MLKL necrosome and further activates macrophage necroptosis. Subsequently, multiple Z-DNA sequences were predicted to be present in the ASFV genome. The Z-DNA signals were further confirmed to be present and colocalized with ZBP1 in the cytoplasm and nucleus of ASFV-infected cells. Moreover, ZBP1-mediated macrophage necroptosis provoked the extracellular release of proinflammatory cytokines, including TNF-α and IL-1ß induced by ASFV infection. Finally, we demonstrated that ZBP1-mediated necroptosis signaling inhibits ASFV replication in host macrophages. Our findings uncovered a novel mechanism by which ASFV induces macrophage necroptosis by facilitating Z-DNA accumulation and ZBP1 necrosome assembly, providing significant insights into the pathogenesis of ASFV infection.

Exosomes in Osteoarthritis: A Review on Their Isolation Techniques and Therapeutic Potential.

Background: Exosomes are the smallest extracellular vesicles (30-150 nm) secreted by all cell types, including synovial fluid. However, because biological fluids are complex, heterogeneous, and contain contaminants, their isolation is difficult and time-consuming. Furthermore, the pathophysiology of osteoarthritis (OA) involves exosomes carrying complex components that cause macrophages to release chemokines and proinflammatory cytokines. This narrative review aims to provide in-depth insights into exosome biology, isolation techniques, role in OA pathophysiology, and potential role in future OA therapeutics. Methods: A literature search was conducted using PubMed, Scopus, and Web of Science databases for studies involving exosomes in the osteoarthritis using keywords "Exosomes" and "Osteoarthritis". Relevant articles in the last 15 years involving both human and animal models were included. Studies involving exosomes in other inflammatory diseases were excluded. Results: Despite some progress, conventional techniques for isolating exosomes remain laborious and difficult, requiring intricate and time-consuming procedures across various body fluids and sample origins. Moreover, exosomes are involved in various physiological processes associated with OA, like cartilage calcification, degradation of osteoarthritic joints, and inflammation. Conclusion: The process of achieving standardization, integration, and high throughput of exosome isolation equipment is challenging and time-consuming. The integration of various methodologies can be employed to effectively address specific issues by leveraging their complementary benefits. Exosomes have the potential to effectively repair damaged cartilage OA, reduce inflammation, and maintain a balance between the formation and breakdown of cartilage matrix, therefore showing promise as a therapeutic option for OA.

Altered ontogeny and transcriptomic signatures of tissue-resident pulmonary interstitial macrophages ameliorate allergic airway hyperresponsiveness.

Introduction: Environmental exposures and experimental manipulations can alter the ontogenetic composition of tissue-resident macrophages. However, the impact of these alterations on subsequent immune responses, particularly in allergic airway diseases, remains poorly understood. This study aims to elucidate the significance of modified macrophage ontogeny resulting from environmental exposures on allergic airway responses to house dust mite (HDM) allergen. Methods: We utilized embryonic lineage labeling to delineate the ontogenetic profile of tissue-resident macrophages at baseline and following the resolution of repeated lipopolysaccharide (LPS)-induced lung injury. We investigated differences in house dust mite (HDM)-induced allergy to assess the influence of macrophage ontogeny on allergic airway responses. Additionally, we employed single-cell RNA sequencing (scRNAseq) and immunofluorescent staining to characterize the pulmonary macrophage composition, associated pathways, and tissue localization. Results: Our findings demonstrate that the ontogeny of homeostatic alveolar and interstitial macrophages is altered after the resolution from repeated LPS-induced lung injury, leading to the replacement of embryonic-derived by bone marrow-derived macrophages. This shift in macrophage ontogeny is associated with reduced HDM-induced allergic airway responses. Through scRNAseq and immunofluorescent staining, we identified a distinct subset of resident-derived interstitial macrophages expressing genes associated with allergic airway diseases, localized adjacent to terminal bronchi, and diminished by prior LPS exposure. Discussion: These results suggest a pivotal role for pulmonary macrophage ontogeny in modulating allergic airway responses. Moreover, our findings highlight the implications of prior environmental exposures in shaping future immune responses and influencing the development of allergies. By elucidating the mechanisms underlying these phenomena, this study provides valuable insights into potential therapeutic targets for allergic airway diseases and avenues for further research into immune modulation and allergic disease prevention.

Deleterious Anti-Inflammatory Macrophage Recruitment in Early Post-Infarction Phase: Unraveling the IL-6/MCP-1/STAT3 Axis.

Using a translational approach with an ST-segment myocardial infarction (STEMI) cohort and mouse model of myocardial infarction, we highlighted the role of the secreted IL-6 and MCP-1 cytokines and the STAT3 pathway in heart macrophage recruitment and activation. Cardiac myocytes secrete IL-6 and MCP-1 in response to hypoxic stress, leading to a recruitment and/or polarization of anti-inflammatory macrophages via the STAT3 pathway. In our preclinical model of myocardial infarction, neutralization of IL-6 and MCP-1 or STAT3 pathway reduced infarct size. Together, our data demonstrate that anti-inflammatory macrophages can be deleterious in the acute phase of STEMI.

Yishen Tongbi decoction attenuates inflammation and bone destruction in rheumatoid arthritis by regulating JAK/STAT3/SOCS3 pathway.

Background: Yishen-Tongbi Decoction (YSTB), a traditional Chinese prescription, has been used to improve syndromes of rheumatoid arthritis (RA) for many years. Previous research has shown that YSTB has anti-inflammatory and analgesic properties. However, the underlying molecular mechanism of the anti-RA effects of YSTB remains unclear. Purpose and study design: The purpose of this research was to investigate how YSTB affected mice with collagen-induced arthritis (CIA) and RAW264.7 cells induced with lipopolysaccharide (LPS). Results: The findings show that YSTB could significantly improve the clinical arthritic symptoms of CIA mice (mitigate paw swelling, arthritis score, thymus and spleen indices, augment body weight), downregulated expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6 and IL-17, while upregulated the level of anti-inflammatory like IL-10 and transforming growth factor-ß (TGF-ß). Meanwhile, YSTB inhibits bone erosion and reduces inflammatory cell infiltration, synovial proliferation, and joint destruction in CIA mice. In addition, we found that YSTB was able to suppress the LPS-induced inflammation of RAW264.7 cells, which was ascribed to the suppression of nitric oxide (NO) production and reactive oxygen species formation (ROS). YSTB also inhibited the production of inducible nitric oxide synthase and reduced the releases of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 in LPS-induced RAW264.7 cells. Furthermore, the phosphorylation expression of JAK2, JAK3, STAT3, p38, ERK and p65 protein could be suppressed by YSTB, while the expression of SOCS3 could be activated. Conclusion: Taken together, YSTB possesses anti-inflammatory and prevention bone destruction effects in RA disease by regulating the JAK/STAT3/SOCS3 signaling pathway.

Progranulin mitigates intestinal injury in a murine model of necrotizing enterocolitis by suppressing M1 macrophage polarization.

Neonatal necrotizing enterocolitis (NEC) is a critical digestive disorder frequently affecting premature infants. Characterized by intestinal inflammation caused by activated M1 macrophages, modulation of macrophage polarization is considered a promising therapeutic strategy for NEC. It has been demonstrated that the growth factor-like protein progranulin (PGRN), which plays roles in a number of physiological and pathological processes, can influence macrophage polarization and exhibit anti-inflammatory characteristics in a number of illnesses. However, its role in NEC is yet to be investigated. Our research showed that the levels of PGRN were markedly elevated in both human and animal models of NEC. PGRN deletion in mice worsens NEC by encouraging M1 polarization of macrophages and escalating intestinal damage and inflammation. Intravenous administration of recombinant PGRN to NEC mice showed significant survival benefits and protective effects, likely due to PGRN's ability to inhibit M1 polarization and reduce the release of pro-inflammatory factors. Our findings shed new light on PGRN's biological role in NEC and demonstrate its potential as a therapeutic target for the disease.

Macrophages and pulmonary fibrosis: a bibliometric and visual analysis of publications from 1990 to 2023.

Background: The role of macrophages in the symptomatic and structural progression of pulmonary fibrosis (PF) has garnered significant scholarly attention in recent years. This study employs a bibliometric approach to examine the present research status and areas of focus regarding the correlation between macrophages and PF, aiming to provide a comprehensive understanding of their relationship. Methodology: The present study employed VOSviewer, CiteSpace, and Microsoft Excel software to visualize and analyze various aspects such as countries, institutions, authors, journals, co-cited literature, keywords, related genes, and diseases. These analyses were conducted using the Web of Science core collection database. Results: A comprehensive collection of 3,479 records pertaining to macrophages and PF from the period of 1990 to 2023 was obtained. Over the years, there has been a consistent increase in research literature on this topic. Notably, the United States and China exhibited the highest level of collaboration in this field. Through careful analysis, the institutions, authors, and prominent journals that hold significant influence within this particular field have been identified as having the highest publication output. The pertinent research primarily concentrates on the domains of Biology and Medicine. The prevailing keywords encompass pulmonary fibrosis, acute lung injury, idiopathic pulmonary fibrosis, and others. Notably, TGFß1, TNF, and CXCL8 emerge as the most frequently studied targets, primarily associated with signaling pathways such as cytokine-cytokine receptor interaction. Additionally, cluster analysis of related diseases reveals their interconnectedness with ailments such as cancer. Conclusion: The present study employed bibliometric methods to investigate the knowledge structure and developmental trends in the realm of macrophage and PF research. The findings shed light on the introduction and research hotspots that facilitate a more comprehensive understanding of macrophages and PF.

Techniques for evaluating the ATP-gated ion channel P2X7 receptor function in macrophages and microglial cells.

Resident macrophages are tissue-specific innate immune cells acting as sentinels, constantly patrolling their assigned tissue to maintain homeostasis, and quickly responding to pathogenic invaders or molecular danger signals molecules when necessary. Adenosine triphosphate (ATP), when released to the extracellular medium, acts as a danger signal through specific purinergic receptors. Interaction of ATP with the purinergic receptor P2X7 activates macrophages and microglial cells in different pathological conditions, triggering inflammation. The highly expressed P2X7 receptor in these cells induces cell membrane permeabilization, inflammasome activation, cell death, and the production of inflammatory mediators, including cytokines and nitrogen and oxygen-reactive species. This review explores the techniques to evaluate the functional and molecular aspects of the P2X7 receptor, particularly in macrophages and microglial cells. Polymerase chain reaction (PCR), Western blotting, and immunocytochemistry or immunohistochemistry are essential for assessing gene and protein expression in these cell types. Evaluation of P2X7 receptor function involves the use of ATP and selective agonists and antagonists and diverse techniques, including electrophysiology, intracellular calcium measurements, ethidium bromide uptake, and propidium iodide cell viability assays. These techniques are crucial for studying the role of P2X7 receptors in immune responses, neuroinflammation, and various pathological conditions. Therefore, a comprehensive understanding of the functional and molecular aspects of the P2X7 receptor in macrophages and microglia is vital for unraveling its involvement in immune modulation and its potential as a therapeutic target. The methodologies presented and discussed herein offer valuable tools for researchers investigating the complexities of P2X7 receptor signaling in innate immune cells in health and disease.