Eosinophils are leukocytes involved in defense against multicellular parasites and other pathogens. Like neutrophils, eosinophils can undergo a cytolytic form of cell death known as eosinophil extracellular trap cell death (EETosis), during which they release DNA and cytoplasmic granules to form eosinophil extracellular traps (EETs). Here, we demonstrate that eosinophil granules released during EETosis exhibit distinct autofluorescence in both purified human eosinophil cultures and mixed leukocyte cultures. This autofluorescence allowed detection of EETosis in mixed leukocyte cultures without additional staining or eosinophil isolation. We further show that 2 vanilloid compounds, previously identified as inhibitors of NETosis, suppress phorbol 12-myristate 13-acetate-induced EETosis. By measuring the autofluorescence intensity of released eosinophil granules, we successfully quantified the inhibitory effects of these compounds on EETosis. Our autofluorescence-based assay enables rapid and convenient detection of EETosis and provides a useful tool for in vitro studies of eosinophil biology.
People living with HIV experience an earlier risk for onset of aging-related chronic inflammatory comorbidities, including cardiovascular disease. We previously showed that the proportion of macrophages was higher in the heart of older rhesus macaques (Macaca mulatta) with normal histopathology as well as in all age groups of macaques with cardiac pathology. In the present study, we investigated the effects of simian immunodeficiency virus (SIV) infection on cell density and macrophages in heart tissues of adult rhesus macaques. Heart tissues were evaluated by hematoxylin and eosin staining and immunofluorescence staining to assess the number and phenotype of heart macrophages that could change with SIV infection. Cardiac cellularity was lower in hearts of SIV-infected young and adult macaques, as well as in uninfected aged macaques, compared with uninfected adult rhesus macaques. This lower cellularity was associated with higher percentages of CD163+ macrophages in hearts of SIV-infected young adult macaques, similar to that of uninfected aged macaques. Higher percentages of CD163+ macrophages were also observed in diseased hearts of both SIV-infected and uninfected adult animals that appeared to be short-lived macrophages, based on 5-bromo-2'-deoxyuridine labeling. In contrast, macrophages retaining in vivo-administered dextran, indicative of long-lived macrophages, predominated among cardiac macrophages in both SIV-infected and uninfected animals of all ages. Together, these results suggest SIV infection induces changes in cardiac cellularity and macrophage composition that resemble those observed during aging. This rhesus macaque model supports continued studies to further dissect the shifting dynamics of macrophage subsets and their role in heart adaptations in response to HIV infection and aging.
Early and rapid identification of sepsis is critical for improving clinical outcomes; however, reliable real-time biomarkers remain unavailable. The scattergram parameters of peripheral blood circulating leukocytes are altered during infection and sepsis. This study aims to explore the cellular biological characteristics underlying these changes, with a particular focus on morphological complexity. An in vitro human whole-blood infection model was constructed by stimulating isolated healthy human peripheral blood with lipopolysaccharide (LPS). Concurrently, a sepsis mouse model was established via intraperitoneal LPS injection. Subsequently, protein kinase C and myeloid differentiation primary-response protein 88 (MyD88) inhibitors were administered separately to establish corresponding inhibition models in mice. The total lysosomal contents in leukocytes and monocytes were then detected to analyze the intrinsic mechanism responsible for the sepsis-associated changes in the scattergram parameters. The results indicated elevated lysosome counts in leukocytes raised N_WBC_SFL_W, whereas those in monocytes drove higher D_Mon_SSC_W under septic conditions. This study reveals that the increased lysosomal content is the primary cause of the heightened number of cytoplasmic granules in leukocytes during sepsis. Furthermore, it clarifies that the characteristic changes in the scattergram parameters N_WBC_SFL_W and D_Mon_SSC_W detected through hematological analysis are a consequence of alterations in the lysosomal content. Collectively, our finding demonstrates that sepsis-induced alterations in leukocytes are hematological manifestations of aberrant lysosomal expansion, providing novel mechanistic insights for sepsis diagnosis and potential therapeutic monitoring.
Multiple myeloma (MM) is a neoplastic disease caused by abnormal clonal plasma cells in the bone marrow (BM). Macrophages (MФs) are essential components of BM microenvironment and crucial mediators of plasma cells. Therefore, investigating alterations in the specific MФ subsets within the MM BM microenvironment is of significant importance for elucidating the underlying mechanisms of MM. Here, using flow cytometry, we characterized the dynamics of BM MФ subsets in newly diagnosed multiple myeloma (NDMM) patients and complete remission multiple myeloma (CRMM) patients. We found that NDMM patients exhibited increased infiltration of CD163+CD206+ MФs and elevated levels of interleukin (IL)-6 and IL-10 in the BM compared with CRMM patients and healthy control subjects. BM intermediate monocytes and nonclassical monocytes were increased in NDMM patients compared with those in CRMM patients and healthy control subjects. Furthermore, we observed upregulated CCR2 and CSF-R expression on the BM CD163+CD206+ MФs. Notably, the BM CD163+CD206+CCR2+ MФs were positively correlated with the increased level of IL-6, while CD163+CD206+CSF-R+ MФs were positively correlated with the level of IL-10. Additionally, clinical correlation analysis revealed that an increased proportion of CD163+CD206+CCR2+ MФs was negatively correlated with hemoglobin levels and positively correlated with β2-microglobulin and lactate dehydrogenase. Moreover, a higher proportion of CD163+CD206+CCR2+ MФs was detected in the BM of MM patients with poor prognosis. Altogether, our findings suggest that higher proportion of BM CD163+CD206+ MФs is involved in pathogenesis of MM, and that CCR2 may be potential therapeutic targets for improving antitumor therapy in MM.
Single-cell RNA sequencing (scRNA-seq) resolves cell types and molecular phenotypes within heterogeneous specimens but typically requires fresh, high-quality single-cell suspensions processed immediately to preserve transcriptional profiles. This constraint complicates samples with long preparation times and prevents collection at remote sites lacking single-cell instrumentation. Several commercial assays now enable preservation at the point of collection through fixation or cryopreservation, allowing processing to occur months later. The Association of Biomolecular Research Facilities' DNA Sequencing and Genomics and Bioinformatics Research Groups undertook a cross-platform, multisite study to assess the performance and reproducibility of three such platforms: 10x Genomics FLEX, Parse Biosciences Evercode WT v2, and Honeycomb Bio HIVE. Total leukocytes and peripheral blood mononuclear cells (PBMCs) were isolated from a single healthy individual, with EasySep reagent used for red blood cell depletion of the leukocyte fraction. Cells were characterized by a 21-color flow cytometry panel to provide a reference, and the remaining material was fixed or cryopreserved according to each platform's protocol. Preserved leukocyte samples were prepared in parallel by two technicians ("A" and "B" replicates) and distributed to multiple ABRF member core facilities for downstream processing, while fresh leukocytes processed with the 10x 3' v3.1 (3pGEX) chemistry served as a reference. Libraries were sequenced at a central site, and performance was evaluated across standard scRNA-seq quality control metrics, gene and transcript detection sensitivity, cell-type discovery and annotation, differential expression, and correlation analyses. Data from all platforms integrated effectively and produced concordant results for cell-type annotation and relative abundance, with cell-type proportions broadly consistent with the flow cytometry reference. However, platform-specific expression signatures were evident for a subset of genes, and cross-site reproducibility varied between methods, with the FLEX workflow showing greater susceptibility to technical variation introduced during on-site sample processing. Preservation-based methods (FLEX and HIVE) showed better retention of fragile granulocyte populations than fresh samples processed with the 10x 3pGEX workflow. Improvements to preservation methods are changing how single-cell research is conducted by decoupling sample collection from downstream processing. Our investigation into the performance and reproducibility of each platform provides a resource to help investigators and core facilities select the most appropriate single-cell preservation workflow given their sample type, cell populations of interest, sample collection logistics, and laboratory infrastructure constraints.
Autologous cell therapy is a patient-embraced approach to inflammatory disease. Murine models demonstrate that alternatively activated (or regulatory) macrophages can reduce the severity of disease, and human macrophages (M0) educated with interleukin-4 (M(IL4)) limit murine colitis. M0 and M(IL4) from healthy donors and individuals with Crohn's disease (active or remission) were assessed by qPCR, characterized by a time-of-flight mass cytometry (CyTOF) comparison of 16 myeloid marker molecules expression, and subtypes of M(IL4)s tested in the murine dinitrobenzene sulfonic acid (DNBS) model of colitis and in vitro co-culture with T cells. Most macrophage preparations responded to IL-4 by increasing CD206, CCL18 and RAMP1 mRNA expression and produced mediators that promoted epithelial repair in a wound assay with the human CaCo2 epithelial line. Single-cell clustering by FlowSOM defined 8 distinct subpopulations (meta-clusters) of macrophages, the proportions of which were unaffected by biological sex, age or cryopreservation: meta-cluster 4 (CD206highPD-L1highHLA-DRhigh) represented 20 to 40% of M0s, with a higher proportion in controls compared with active Crohn's disease. IL-4 treatment significantly expanded meta-cluster 4 in all M0s. Transfer of M(IL4)-CD206high (ie, predominantly meta-cluster 4) into rag1-/- mice significantly reduced DNBS-induced colitis, and to a greater extent than M(IL4)-CD206low from the same individual. IL-10 production was increased in M(IL4)-CD206high-T-cell co-cultures. Having confirmed the prohealing effect of the human M(IL4), the predominant meta-cluster M(IL4)-CD206high was found to have superior anticolitic effect. Reduced numbers of M0-CD206high and M(IL4)-CD206high cells in some individuals with Crohn's disease suggest that their absence may contribute to more severe inflammation or reduced healing capacity.
Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disorder of the gut, whose pathogenesis is closely related to immune dysregulation. Regulatory T cells (Tregs), as a key cell population maintaining intestinal immune tolerance, exhibit not only reduced frequency but, more critically, profound functional deficiencies, including the generation of proinflammatory ex-Tregs, and loss of lineage stability in the inflamed gut microenvironment of IBD. This review systematically delineates the sophisticated biological characteristics of Tregs, with a particular focus on the epigenetic and metabolic checkpoints that govern their stability. We critically summarize the aberrant changes of Tregs in IBD, emphasizing mechanisms such as inflammatory cytokine-induced Treg plasticity (eg Th1-like or Th17-like conversion) and microbiota-metabolite axis-mediated functional modulation. Furthermore, we provide an in-depth analysis of emerging therapeutic strategies aimed at restoring Treg suppressive capacity, including selective cytokine therapy (eg low-dose/engineered IL-2), adoptive transfer of engineered antigen-specific Tregs (including CAR-Tregs), microbiome/dietary interventions, and pharmacological modulation of Treg differentiation. We also discuss the disrupted crosstalk between Tregs and other innate and adaptive immune cells within the IBD milieu. Finally, this review highlights how cutting-edge technologies like single-cell multiomics and spatial transcriptomics are delineating the heterogeneous landscape and uncovering novel, disease-specific Treg subpopulations, thereby paving the way for precise Treg-targeting therapies. Unlike previous reviews that broadly catalog Treg abnormalities, this review offers a refined conceptual framework centered on the critical distinction between lineage-defective ex-Tregs and exhausted effector Tregs, and integrates recent multiomics insights to redefine Treg functional failure in IBD.
Excess neutrophil apoptosis and the release of neutrophil extracellular traps (NETs) in systemic lupus erythematosus (SLE) lead to the accumulation of cell debris and the production of auto-antibodies targeting nuclear proteins and DNA. SLE neutrophil activation is regulated by changes in gene expression, notably expression of type I interferon-response genes and genes coding for granule proteins. This observational study measured both mRNA and small noncoding RNAs in SLE (n = 11) and healthy control (HC, n = 10) ultra-pure blood neutrophils to identify changes in expression that are involved in regulating neutrophil phenotype. Using RNAseq, we identified significant differential expression (DE) of 69 microRNAs, 63 other small noncoding RNAs, 236 piwiRNAs, and 83 tRNA fragments in SLE neutrophils compared to HC (false discovery rate [FDR] adj. P < 0.05). We also identified 78 significant alternative splicing events across 64 genes (FDR adj. P < 0.05, Δpercent spliced in (PSI) > 0.1 or < -0.1). Bioinformatic analysis of miRNA:mRNA DE genes predicted significant activation of autophagy, neutrophil degranulation, interferon alpha/beta signaling, and apoptosis pathways in SLE neutrophils. Translation and mRNA processing were predicted to be downregulated. microRNAs implicated in NETs production were miR-155-5p, miR-146a-5p, and miR-let-7b-5p (FDR adj. P < 0.05). SNORD89 was identified as a potential promoter of apoptosis in SLE neutrophils, along with alternative splicing of apoptosis genes myeloid cell leukemia-1 (MCL1), caspase-8 (CASP8), and death-associated protein kinase-2 (DAPK2) (FDR adj. P < 0.05). Our study, for the first time, describes dysregulated expression of small noncoding RNAs in SLE neutrophils and proposes noncoding RNA and alternative gene splicing as regulators of neutrophil-driven disease pathology in SLE.
Eosinophils are highly granulated white blood cells first identified as "granule blood cells" in the 19th century. In this review, we discuss techniques used to identify these unique cells and explore how they release a range of pro- and anti-inflammatory mediators. They are easily detected with a range of cellular dyes due to highly cationic proteins stored within their crystalloid granules. Cationic proteins include major basic protein (MBP), eosinophil peroxidase (EPX), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and Charcot-Leyden crystal protein (CLC, also known as galectin-10) that serve as potent antimicrobial factors and possess a high affinity for negatively charged molecules including eosin and fluorescein isothiocyanate (FITC). Crystalloid granules are responsible for autofluorescence and nonspecific binding to both fluorophores and antibodies during immunolabeling for fluorescence microscopy, flow cytometry, CyTOF, and other antibody-based detection methods. Eosinophils release a plethora of mediators that have roles in immunity and homeostasis. Here, we describe six different categories of mediators released by eosinophils and their analyses: (1) cationic granule proteins (MBP, EPX, EDN, ECP, and CLC), (2) cytokines and chemokines, (3) reactive oxygen species, (4) eicosanoid production, (5) eosinophil extracellular trap formation, and (6) exosomes. We also describe novel transcriptional markers where new subtypes of eosinophils are characterized through the development of single cell RNA sequencing, showing additional transcripts appearing in eosinophils from patients with diseases. Future work on eosinophils is anticipated to lead to a greater understanding of their role in immunity and diseases based on novel emerging techniques.
Both in clinical practice and translational research, cell differentiation of leukocytes provides important diagnostic information and insights into pathophysiological mechanisms. The current gold-standard method for bronchoalveolar lavage fluid (BALF) analysis involves histochemical staining of cytospins, followed by manual morphological quantification. This approach however is labor-intensive, time-consuming, and highly operator-dependent, limiting its efficiency and throughput. This study proposes a deep learning framework for rapid, automated 3D leukocyte differentiation using label-free higher harmonic generation microscopy (HHGM). 3D leukocyte imaging was performed with label-free HHGM in a few minutes. Two deep learning models, ResNet 3D-50 and Vision Transformer (ViT) 3D, were trained, validated and tested for leucocyte differentiation on both BALF and blood fraction samples from 16 interstitial lung disease (ILDs) and 19 acute respiratory distress syndrome (ARDS) patients. Deep-learning model-prediction and cytospin analysis were performed by separate investigators. The results were compared using Bland-Altman analysis. The proposed framework achieved accuracies above 86% for BALF and above 96% for blood samples under five-fold cross-validation. The approach shows close agreement with gold-standard cytological analyses, with mean differences of <5% across leukocyte subpopulations. By integrating the label-free imaging capabilities of HHGM with deep learning, this study established a fast, accurate and high-throughput leukocyte differentiation in fresh BALF and blood samples. By significantly improving efficiency and reproducibility, this technology has the potential to transform clinical workflows and advance precision medicine.
Eosinophils, cells of the innate immune system, have a unique architecture, which includes morphologically complex secretory granules and a prominent, activation-responsive vesiculotubular system. In human eosinophils, this system is collectively termed eosinophil sombrero vesicles (EoSVs) because of its peculiar morphology feasibly identifiable by transmission electron microscopy. Considered as "microgranules" or "small granules" in the past, EoSVs are currently defined as active components of the eosinophil secretory pathway, sprouting from specific granules and trafficking granule-derived products in the eosinophil cytoplasm. Eosinophil secretory proteins associated with EoSVs include cationic proteins, cytokines, cytokine receptors, membrane fusion complexes, and tetraspanins. Beyond the intracellular landscape, emerging evidence has revealed a more complex functional repertoire for EoSVs. This review offers an overview of EoSVs as key participants in the mechanisms of eosinophil secretion during immune responses, incorporating the recent understanding of these transport carriers as extracellular vesicles, potentially implicated in the traffic of eosinophil products in the extracellular medium and intercellular communication.
Background and Objectives: Oxidative stress is widely recognized as a key contributor to both the development and progression of chronic obstructive pulmonary disease (COPD), particularly during acute exacerbations (AECOPD). 8-Hydroxy-2'-deoxyguanosine (8-OHdG), a marker of oxidative DNA damage, has been insufficiently investigated as a systemic biomarker in this setting. This study evaluated the relationship between serum 8-OHdG levels, airflow limitation severity, and smoking status in patients hospitalized with AECOPD. Materials and Methods: We conducted a cross-sectional study including 176 patients admitted for AECOPD and stratified according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages. The study population comprised patients with comparable distributions of age, sex, smoking status, diabetes, cardiovascular comorbidities, and area of residence across GOLD categories. Serum 8-OHdG, leukocyte count, neutrophil percentage, C-reactive protein (CRP), fibrinogen, and procalcitonin were measured. Group comparisons, multivariable logistic regression analyses, and smoking-status subgroup analyses were performed. Results: Serum 8-OHdG levels increased significantly with advancing airflow limitation severity (p = 0.038), with higher concentrations observed in patients with GOLD 4 disease compared to GOLD 1-2 disease (p = 0.023). In multivariable analysis, 8-OHdG was the only biomarker independently associated with GOLD stage (OR = 2.44, 95% CI: 1.12-5.31, p = 0.025). Higher serum 8-OHdG levels were also independently associated with smoking status (OR = 1.20, 95% CI: 1.05-1.37, p = 0.022). Ever-smokers demonstrated significantly higher 8-OHdG concentrations than never-smokers in GOLD 1-2 and GOLD 3 disease, whereas procalcitonin levels were higher among never-smokers with advanced COPD. Conclusions: Serum 8-OHdG levels measured during AECOPD are associated with both airflow limitation severity and smoking status. These findings support the potential role of oxidative DNA damage as a clinically relevant component of COPD pathophysiology and suggest that serum 8-OHdG may represent a useful biomarker for disease characterization in patients experiencing acute exacerbations.
As tissue-resident innate immune cells of the central nervous system, microglia are capable of acquiring innate immune memory-a persistent state of functional reprogramming triggered by prior stimuli. This memory typically manifests as 3 distinct phenotypes: trained immunity, immune tolerance, and immune exhaustion. In this review, we synthesize current knowledge on the metabolic and epigenetic mechanisms that govern these 3 forms of microglial innate immune memory. We further summarize and discuss how each phenotype is induced in microglia and its respective pathophysiological roles in neurological disorders. Owing to their slow turnover and unique tissue-resident characteristics, microglia sustain long-lasting memory states that can profoundly influence the trajectory of neuroinflammation and neurodegeneration. Finally, we highlight the bidirectional effects of microglial immune memory on disease progression, discuss emerging therapeutic strategies aimed at modulating these memory states, and outline key translational challenges that remain to be addressed.
Malaria caused by Plasmodium falciparum remains a leading cause of childhood mortality in sub-Saharan Africa. The intraerythrocytic parasite can evade complement-mediated destruction through recruitment of human Factor H (FH). In vitro, Factor H-related protein 1 (FHR-1) competes with FH for binding sites, suggesting a potential role in modulating complement activation during infection. Because one-third of Africans carry a CFHR3/1 deletion, we examined the impact of FHR-1 deficiency on malaria severity. We analyzed plasma from 500 Ghanaian children presenting with different clinical manifestations. One-third of plasma samples lacked detectable FHR-1 protein, but logistic regression analysis revealed no association between FHR-1 deficiency and risk of severe malaria. In contrast, FHR-1 deficiency was overrepresented among children presenting with anemia and multiple syndromes and affected children exhibited markedly lower hemoglobin levels. These findings indicate that FHR-1 deficiency does not affect overall progression of severe falciparum malaria, but correlates with severe malarial anemia.
Idiopathic inflammatory myopathies are a heterogeneous group of autoimmune disorders characterized by skeletal muscle inflammation and systemic immune activation. Although traditionally viewed as T cell-driven diseases, recent evidence suggests that B lymphocytes may also contribute to disease mechanisms through both antibody-dependent and noncanonical pathways. Beyond their role in autoantibody production, B cells might influence immune regulation by modulating cytokine networks, presenting antigens, and interacting with T cells and stromal elements within inflamed tissues. These findings have prompted renewed interest in understanding B cell heterogeneity and its possible impact on the initiation and maintenance of chronic inflammation in idiopathic inflammatory myopathies. However, the exact contribution of these cells to tissue damage and clinical variability remains uncertain. Advances in high-dimensional cytometry, transcriptomics, and tissue profiling are beginning to delineate distinct B cell signatures associated with disease activity, yet the causal links to pathogenesis remain unclear. The observed clinical benefit of B cell-depleting therapies, though variable among patients, reinforces their potential relevance while underscoring the complexity of immune interactions in idiopathic inflammatory myopathies. This review aims to synthesize current knowledge on B cells in idiopathic inflammatory myopathies and to discuss how emerging mechanistic insights could refine our understanding of disease heterogeneity and guide future therapeutic approaches.
Interleukin-32 is recognized as a potent proinflammatory mediator in various infectious contexts; however, its precise contribution to host defense against Streptococcus pneumoniae (S.pn) remains to be fully elucidated. This study demonstrates that S.pn challenge robustly upregulates interleukin-32 expression in human peripheral blood mononuclear cells and THP-1 cells. Functional assays reveal that exogenous recombinant human interleukin-32γ significantly potentiates the phagocytic activity of both murine (RAW 264.7) and human (THP-1-derived) macrophages against S.pn. Mechanistically, recombinant human interleukin-32γ-induced phagocytosis is mediated by the upregulation and secretion of soluble epoxide hydrolase (sEH), a process contingent upon the activation of the nuclear factor κB (NF-κB) signaling pathway. Furthermore, pharmacological inhibition of either NF-κB or sEH effectively abrogates the prophagocytic effects of recombinant human interleukin-32γ, confirming the functional requirement of the NF-κB-sEH axis in this response. Collectively, these results elucidate a novel interleukin-32γ-NF-κB-sEH regulatory cascade that facilitates macrophage-mediated bacterial clearance, thereby identifying a potential therapeutic target for the management of pneumococcal infections.
The proliferation, differentiation, and survival of cells of the macrophage lineage depends on signals from the macrophage colony-stimulating factor receptor (CSF1R). On a C57BL/6J background homozygous kinase-dead Csf1r mutation (Csf1rE631K/E631K-E631Km/m) causes perinatal lethality. By contrast, E631Km/m mice on a mixed genetic background (C57 × BALB/c) were osteoclast-deficient/osteopetrotic and growth retarded but did not develop hydrocephalus or other major structural brain abnormalities seen in inbred Csf1r-/- mice, and they were viable as adults. Outbred E631Km/m mice lacked tissue-resident macrophages detected with a Csf1r-EGFP transgene or F4/80 in most major organs, including the brain, but aside from reproductive organs, which were underdeveloped, no gross histological abnormalities were observed. E631Km/m genotype was associated with reduced blood monocytes, the loss of circulating C1Q, reduced IGF1, and increased CSF1. In mice, F4/80+/CD169+ resident marrow macrophages are believed to be essential components of erythroblastic and hematopoietic island niches. These cells were undetectable in E631Km/m marrow. Their absence was associated with granulocytosis and B-cell deficiency in marrow, blood, and spleen, whereas the relative abundance of pluripotent and committed progenitors was not affected. Erythropoietic homeostasis was also maintained; erythroblasts were physically associated with a minor residual population of CSF1R-independent CD169-/F4/80+ marrow macrophages. We conclude that many developmental and homeostatic functions attributed to mouse resident-tissue macrophages are redundant, species-specific, or restricted to inbred strains.
Macrophages coordinate cytokine responses by integrating signals from pattern recognition receptors including NOD2 and TLR4. NOD2 detects muramyl dipeptide derived from bacterial peptidoglycan, and TLR4 recognizes lipopolysaccharide in the outer membrane of Gram-negative bacteria. NOD2 and TLR4 signals synergize to enhance cytokine production in myeloid cells. While synergy can support host defense, it must be regulated to minimize the risk of excessive inflammation. The lipid phosphatase SHIP, a negative regulator of Class I PI3Ks, reduces inflammatory signaling, but its role during NOD2-TLR4 co-stimulation remains undefined. We found that SHIP limits IL-1β production by blocking synergy in bone marrow-derived macrophages co-stimulated with muramyl dipeptide and lipopolysaccharide. SHIP-/- macrophages showed a synergistic increase in IL-1β that was not evident in SHIP+/+ macrophages. Moreover, reducing SHIP protein concentrations by differentiation in different growth factors, IL-4 treatment, or siRNAs enabled synergy for IL-1β production in SHIP+/+ macrophages. Pharmacologic inhibition of SHIP's catalytic activity did not promote synergy, and similarly, blocking PI3K had no effect, suggesting that the response is independent of SHIP's phosphatase activity. Synergy for IL-1β production was dependent on NOD2 signaling despite NOD2 stimulation alone resulting in little to no IL-1β. Moreover, IL-1β was selectively enhanced in SHIP-/- macrophages during co-stimulation with MDP and LPS, or when MDP stimulation preceded LPS. These findings identify SHIP's adaptor function as a negative regulator of IL-1β induced by NOD2-TLR4 co-stimulation in macrophages, and suggest that SHIP acts as a gatekeeper for macrophage IL-1β during early innate immune activation.
Cold atmospheric plasma (CAP) has garnered substantial attention in biomedical science, owing to its wide therapeutic applications in wound healing, disinfection, dentistry, cancer care, and inflammation. The anti-inflammatory effects of CAP have been studied, but, to our knowledge, there are no reports investigating its effect on T cell hyperactivation-associated pathologies. This study examines CAP's impact on antigen-driven and homeostatic T cell proliferation and its potential to prevent acute graft-vs-host disease (GvHD). CAP treatment significantly attenuated GvHD-associated mortality and morbidity in mice. CAP inhibited stimulation-induced T cell activation, surface marker expression, cytokine secretion, and proliferation, without inducing cell death, indicating noncytotoxic immunomodulation. CAP modulated cellular redox, and pretreatment with N-acetylcysteine- or PEGylated catalase-abrogated CAP-mediated suppression of mitogen-induced T cell responses. Moreover, CAP-mediated inhibition of stimulation-induced T cell responses was associated with suppression of the immune-regulatory, redox-sensitive transcription factors NF-κB and Nrf2. These findings underscore immunotherapeutic potential of CAP in treatment of disorders linked to T cell hyperactivation.
Natural killer (NK) cells are central to cancer immunosurveillance and immunotherapy. Their ability to engage in killing is critical for efficient target elimination and depends on tightly regulated Ca2+ signaling controlling granzyme degranulation. Toll-like receptor (TLR) engagement has been linked to Ca2+ signaling in other immune cell types, while a specific role in NK cells remains unresolved. Given that NK cells express a broad range of TLRs, and that presence of TLR ligands-including damage- and pathogen-associated molecular patterns-can influence clinical scenarios including adoptive NK cell therapy, investigating TLR-driven Ca2+ signaling in NK cells is particularly relevant. Here, we examined how stimulation with selected TLR ligands influences Ca2+ signaling and NK cell activity. Immediate stimulation induced a rapid elevation of cytosolic Ca2+ in expanded NK cells. Functionally, TLR stimulation increased degranulation and enhanced cytotoxicity at high effector-to-target ratios. Under conditions of target excess, however, TLR-treated NK cells displayed impaired killing, likely due to unbalanced Ca2+ levels. These findings demonstrate that TLR signaling directly modulates Ca2+ flux in NK cells and can either potentiate or impair cytotoxic activity depending on context. Although clinical implications remain hypothetical, such TLR-driven dysregulation affects NK cell killing activity in the inflammatory or pathogen-rich environments frequently encountered after chemotherapy. Our findings suggest that TLR-Ca2+ impact on cytotoxicity should be considered in adoptive transfer therapies where previous treatment affects systemic levels of TLR ligands, such as in patients with acute myeloid leukemia, where NK cell immunotherapy is frequently tested in trials.