As a scientist, teacher, research mentor, and mom, I asked myself, "what is my legacy?" Legacies usually have numbers associated with them. When asked this question in the past, I summed my accomplishments in terms of who, not what. First and foremost, I have my daughter, Cathy, and then scores of spectacular lab trainees (N > 130), many of whom have contributed to the tally of "lab babies." At present, N = 79 lab babies (Cathy, of course, being lab baby #1; there is also 1 lab "grandchild"). Together, we are the "Kovacs Empire." Watching the trainees develop into clinicians/scientists/parents/etc has been among the greatest joys of my career and seeing their kids grow up is an added bonus. Of course, other numbers could be thrown into the mix. For example, I have published >300 papers and have been funded by NIH for >29 consecutive years (along with multiple local, national, other federal funding agencies) and, importantly, have helped launch the careers of many mentees from my lab and elsewhere. The "Kovacs Babies in the Empire" email list is broad and gives me an excuse to check in with the group on a relatively frequent basis. The other part of my "legacy," of course, is the money I have earned. Sorry to the lab members and readers who expect a cut of this, it all goes to Cathy.
Myeloid differentiation primary-response protein 88 (MyD88), an adapter protein used by many Toll-like receptors (TLRs), plays a critical role in initiating inflammatory responses. Our laboratory has previously shown that alcohol intoxication combined with burn injury causes intestinal tissue damage and leakiness. This is accompanied by increased intestinal neutrophil infiltration along with bacterial abundance and translocation. An overgrowth of bacteria can activate TLRs on intestinal epithelial cells (IECs), promoting inflammation and subsequent gut tissue damage. To examine the role of MyD88 in intestine inflammation/leakiness following ethanol and burn injury, we used MyD88-/- mice. MyD88 deficiency in mice prevented the increases in IL-6 and KC levels, MPO activity, as well as the expression of neutrophil mediators Lipocalin2, S100a8, and S100a9 in the intestine. It also attenuated neutrophil extracellular trap (NET) formation and release of superoxide anion (O2-). In addition, MyD88 deficiency improved tight junction; namely ZO-1, Occludin, Claudin 4 and Claudin 8 expression. Finally, using IEC specific MyD88-/-IEC mice, we further found that MyD88 deficiency mitigated intestinal permeability in ethanol and burn. Together, these findings suggest that MyD88 plays a critical role in intestinal inflammation and barrier leakiness following ethanol and burn injury, and therefore, targeting the MyD88 pathway may have therapeutic potential for intervention in burn injury.
Inflammation is a multifaceted response involving molecular and cellular components triggered by diverse stimuli, including physical, chemical, and biological factors. The connection between these stimuli and inflammatory cellular responses involves numerous interactions among molecular components, resulting in cell outputs that can either help resolve potential harmful outcomes or amplify them, leading to further complications. Notably, heat shock factor 1 (HSF1), a transcription factor primarily associated with cellular responses to elevated temperatures, has also been reported to play a key role in modulating inflammation. Indeed, growing evidence suggests a close relationship between HSF1 and intracellular signaling pathways that regulate the secretion of inflammatory cytokines, impacting both infectious and non-infectious diseases. Furthermore, chaperone proteins known as heat shock proteins (Hsps), which HSF1 transcriptionally regulates, also play important roles during inflammation by modulating numerous host factors involved in this process. Here, we examine the interplay between HSF1 and Hsps in the context of inflammation caused by infectious and non-infectious diseases. We also discuss the potential benefits of targeting these factors as strategies for reducing the effects of exacerbated or damaging inflammation.
Myeloid cells orchestrate vascular inflammation through transcriptional programs that control their maturation, effector function, and survival. While lineage-determining transcription factors establish myeloid identity, our understanding of the transcriptional regulation of myeloid cell behavior in chronic inflammatory contexts remains limited. Nuclear factor-Y (NF-Y) is a trimeric CCAAT-binding transcription factor that regulates cell proliferation and differentiation and is essential for maintaining stem and progenitor cell fitness. Here, we investigate NF-Y activity in myeloid cell function and survival using integrated single-cell transcriptomics and myeloid-specific deletion of the NF-YA subunit. At RNA level, NF-Y subunit transcripts were detected across myeloid compartments, with NF-YA enriched in proliferative macrophages and immature neutrophils. In mouse atherosclerotic lesions, low NF-YA levels were associated with macrophages exhibiting lipid-handling and phagocytic signatures and with neutrophils displaying a proinflammatory phenotype. NF-Y deficiency impaired neutrophil adhesion and rolling, increased their susceptibility to apoptosis, and promoted activation and lipid accumulation in macrophages. In vivo, myeloid NF-YA deficiency reduced circulating neutrophil counts, increased macrophage and neutrophil apoptosis during acute inflammation, expanded necrotic cores, leading to larger and more unstable atherosclerotic lesions, and aggravated both atherosclerosis and injury-induced neointimal thickening. These findings identify NF-Y as a transcriptional safeguard of myeloid cell survival during inflammatory stress, thereby shaping disease progression and outcomes in vascular disease.
NK cells effectively eliminate infected cells or tumour cells. The acquisition of cytotoxic functions occurs throughout NK cell functional maturation, a process that is commonly defined by the sequential expression of CD27 and CD11b. In addition, NK cell cytotoxicity is finely tuned by the process of education, which is dependent on MHC I. Expression of effector molecules is not impaired in NK cells from MHC I-deficient mice, but their cytotoxic activity towards target cells is reduced. While the role of MHC I on NK cell education has been extensively studied, its impact on NK cell functional maturation has not been defined. Using two distinct MHC I-deficient mouse models, we confirm that MHC I is not required for expression of effector molecules by NK cells. However, based on CD27 and CD11b expression, we find that fewer NK cells reach the final stage of NK cell functional maturation. Moreover, in β2m-deficient mice, early-stage NK cells express higher levels of granzyme B. Together, these findings demonstrate that NK cell functional maturation is dependent on MHC I and that the expression of effector proteins can be uncoupled from the NK cell functional maturation stages.
Methylation of histone (H) 3 lysine (K) 4 has a well-established role in innate immune responses, but the contribution of H3K4 methyltransferases Kmt2c and Kmt2d in innate immunity is incompletely understood. Using conditional knockout (KO) mouse models, we investigated how Kmt2c- and Kmt2d-deficiencies affect innate immune cell function. Through functional, transcriptomic, and metabolic analyses, we delineate the consequences of disrupted epigenetic regulation on macrophage biology. Our findings reveal that loss of Kmt2c or Kmt2d in macrophages leads to impaired pro-inflammatory cytokine response and phagocytotic capacity, as well as skewed energy metabolism towards glycolysis, highlighting the critical role of H3K4 methylation-dependent chromatin regulation in shaping innate immune cell behavior. This study provides the first comprehensive characterization of innate immune system dysfunction in mouse models with conditional Kmt2c and Kmt2d deletions and offers mechanistic insight into how epigenetic regulators control fundamental immune processes.
Obesity is a disease associated with an increased risk of various medical complications, including cardiovascular problems, diabetes, and infectious diseases. The higher incidence of infections in individuals with obesity is likely due to impaired immune functions. Neutrophils deploy microbicidal functions. However, it is unclear whether neutrophils in individuals with obesity function effectively against infections. To evaluate whether neutrophils in obesity have dysfunctional antimicrobial functions, neutrophils from individuals with normal weight and from individuals with obesity were isolated, and their phenotype and microbicidal functions were assessed. Neutrophils from individuals with obesity had higher expression of CD66b, CD11b, CD15, CD14, CD18, and CD182 on their membranes. The higher expression of these molecules correlated with higher body mass index, suggesting that obesity is associated with neutrophil activation. Accordingly, the phorbol 12-myristate 13-acetate (PMA)-induced respiratory burst in neutrophils from individuals with obesity was greater than in those from individuals without obesity, whereas phagocytosis and PMA-induced NETosis were comparable between neutrophils from individuals with or without obesity, implying that neutrophil functions are not impaired by obesity. However, neutrophil extracellular trap formation of neutrophils from individuals with obesity was selectively reduced in response to Staphylococcus aureus. Nevertheless, the actual killing of these bacteria was similar between neutrophils from individuals with or without obesity. In contrast, neutrophils from individuals with obesity exhibited impaired killing of Pseudomonas aeruginosa and of Klebsiella quasipneumoniae. Together, these findings suggest obesity induces neutrophil activation but disrupts pathogen-specific antimicrobial mechanisms of neutrophils. This deficiency in pathogen clearance could explain the increased susceptibility to infections observed in individuals with obesity.
Patrolling monocytes (pMos) scavenge debris from vessel walls and mediate antibody-dependent cellular phagocytosis, making them attractive cell therapy candidates for cancer, atherosclerosis, and Alzheimer's disease. However, methods to generate pMos for cell therapy are not available. To address this, we developed a 2-step procedure to produce pMos in vitro from murine bone marrow (BM). First, myeloid progenitors were expanded and enriched from BM using cytokines for 4 d. Second, expanded progenitors were differentiated into pMos on delta-like ligand 1 (DLL1)-expressing monolayers for 8 d. We confirmed that in vitro grown pMos expressed the transcription factor Nr4a1 (Nur77) and other canonical pMos surface proteins, and depended on Notch signaling for their development. RNA-sequencing revealed that in vitro pMos expressed hallmark pMos genes, including Cx3cr1, Itgax (CD11c), CD43, Fcγr4, and Cd274 (PD-L1), and their gene signatures clustered closely with in vivo blood and BM pMos. Transcriptomic and phenotypic analyses further demonstrated that in vitro pMos were distinct from classical BM macrophages. Phagocytosis assays demonstrated the function of in vitro pMos in cancer cell uptake. Adoptive transfer studies demonstrated that in vitro pMos persisted within the circulation and lung vasculature during the early post-transfer period compared with BM-derived macrophages, consistent with the vascular-patrolling properties of pMos. Adoptive transfer of pMos reduced lung tumor burden in a metastatic model, supporting an anti-tumoral role for pMos and their ability to mediate immune surveillance in vivo. These findings demonstrate that the DLL1 culture system allows for propagation of functional pMos, enabling studies of pMos biology and their therapeutic potential.
The complement system constitutes a fundamental element of the innate immune response, playing a pivotal role in the identification and eradication of pathogens. The orchestration of complement component activation is closely linked to bone remodeling and the maintenance of skeletal homeostasis. C3a and C5a are notably influential in modulating the differentiation and activity of bone remodeling cells-including osteoclasts, osteoblasts, and osteocytes. Elevated expression or excessive activation of complement components is recognized as a major contributor to bone injury in a variety of inflammatory skeletal disorders. The therapeutic role of complement factors has been evaluated in several models of skeletal diseases. A comprehensive understanding of the complement system in bone remodeling and its therapeutic implications may help in designing innovative strategies to prevent bone degradation and disorders. This review discusses the relationship between the complement system, bone homeostasis, and bone-associated inflammatory diseases. Furthermore, we examine the complement system's potential and its limitations in complement-targeted therapies.
Critical care research focuses on life-threatening conditions such as sepsis, trauma, hemorrhage, and burn injury, which account for millions of hospitalizations and hundreds of thousands of deaths annually in the United States alone. Recent policy initiatives by the US Food and Drug Administration and the National Institutes of Health have promoted new approach methodologies, including organoids, organ-on-chip platforms, and computational models, as alternatives to animal research. While new approach methodologies offer valuable tools for mechanistic investigation and screening applications, this review examines whether current new approach methodology technologies can adequately replace animal models in critical care research. Critical illness involves the whole organism, including dynamic organ-organ interactions, immune-microbiome crosstalk, and adaptive systemic feedback loops. By examining major domains in critical care research and targeted organ injuries, it becomes clear that while new approach methodologies excel at interrogating isolated subsystems, they cannot currently replicate integrated physiological responses. Animal models remain essential for questions requiring assessment of multiorgan dysfunction, therapeutic safety evaluation, and clinically relevant disease trajectories. Premature policy shifts away from animal research will impede advances in critical care medicine. This review proposes a "methodological pluralism" approach that integrates new approach methodologies with appropriately designed animal studies through harmonized endpoints and reverse-translation frameworks. Recommendations include continued refinement of animal models to better represent the heterogeneity of human populations, adoption of quality standards for preclinical research, and strategic deployment of both new approach methodologies and animal models based on fit-for-purpose criteria. We believe this approach will satisfy ethical considerations, scientific rigor, and public health needs in critical care research.
Emerging evidence has highlighted the influence of cellular metabolism on both cancer and T cell growth and survival, including in the context of adoptive cell immunotherapy. It is known that T cells heavily rely on glycolysis for cell proliferation and mediating effector functions. However, less is known about the role of the pentose phosphate pathway (PPP), a parallel metabolic pathway that produces antioxidant and biosynthetic precursors, toward regulating antitumor T cell function. In this review, we summarize the PPP's role as a time-dependent regulator of T cell activation and redox balance, and its association with improved functional capacity in exhausted T cells. We further highlight the contrasting role of the PPP for CD8+ and CD4+ T cell differentiation in guiding antitumor responses, as well as a unique connection in the glycogen-PPP axis to CD8+ memory T cells. Finally, we summarize global metabolic regulators that drive expression of PPP enzymes and discuss future avenues for immunotherapy that could take advantage of our current understanding of the PPP's role in T cell metabolism.
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.
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.
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.
Chronic lymphocytic leukemia (CLL) is a B cell malignancy driven by aberrant signaling and microenvironmental support. Cytoskeletal remodeling contributes to these mechanisms, where the hematopoietic protein HS1 appears to play a key role. Thus, we generated C57BL/6 HS1 knockout (HS1KO) mice to assess its impact on lymphocyte development. Young HS1KO mice showed some alterations in B cell maturation and T cell differentiation, which appeared to normalize with age. In double-transgenic Eμ-TCL1-HS1KO mice, combining HS1 loss with the standard Eμ-TCL1 CLL mouse model, we observed a trend of reduction in the CD4/CD8 ratio, and disrupted splenic architecture with loss of follicular organization. Transcriptomic and kinase profiling of the human CLL cell line MEC1, in which HS1 was knocked out (MEC1-HS1KO), revealed a coherent impairment in terms of cell mobility as highlighted by severely impaired migration in 2D and 3D environments, reduced chemotactic responses to CXCL12 and CCL19; deregulation of cytoskeletal, adhesion, and apoptotic pathways; and increased resistance to apoptosis. We posited that these defects could reduce sensitivity to BTK (Bruton's tyrosine kinase) inhibition, a standard therapeutic approach in CLL. Primary CLL cells from the spleen of Eμ-TCL1-HS1KO mice appeared to show reduced sensitivity to ibrutinib, a finding that was also observed in adoptive transfer experiments designed to equalize disease burden. Wild-type mice receiving Eμ-TCL1-HS1KO leukemic cells tend to be more resistant to ibrutinib, particularly in the peripheral blood and spleen. All together, these findings establish HS1 as a potential regulator linking B cell receptor signaling, cytoskeletal remodeling, leukemic progression, and response to therapy, supporting its potential as a biomarker for BTK inhibitor sensitivity.
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.
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.
Premature birth and cesarean section are associated with increased morbidity and inflammatory diseases. However, their impact on neonatal immunity remains incompletely defined. To explore how gestational age and mode of delivery contribute to early immune programming, we analyzed CD4+ T cells, central regulators of adaptive responses, from preterm neonates and full-term neonates born by cesarean section or vaginal delivery. We performed transcriptomic profiling (mRNA-seq) and functional assessment of T cell activation, proliferation, and cytokine production following stimulation. The mode of delivery emerged as a key factor for CD4+ T cell transcriptome and function. CD4+ T cells from full-term neonates born by vaginal delivery exhibited an immune activation signature, produced higher levels of multiple cytokines, and showed reduced proliferative capacity. In contrast, prematurity was associated with modest changes in basal gene expression relative to full-term cesarean section neonates. CD4+ T cells from preterm neonates displayed enhanced proliferation and increased secretion of inflammatory cytokines (IL-13, TNFα, IL-6, and IL-17F) upon stimulation, consistent with heightened responsiveness. Collectively, our findings show that CD4+ T cells from preterm neonates exhibit augmented inflammatory potential, which becomes more regulated at term. Mode of delivery further contributes to this developmental trajectory: cesarean section is associated with a restrained functional profile, whereas vaginal delivery is associated with a mild immune activation signature and increased responsiveness. These results support a model in which neonatal CD4+ T cell trajectories are established during fetal life and further modulated at birth, highlighting the layered influence of perinatal factors on immune development.
Therapeutic modulation of sepsis-induced immune dysfunction by targeting lymphocyte dysfunction with recombinant IL-7 (rIL-7) and anti-PD-1 (e.g. nivolumab) has shown promise in preclinical and early clinical studies. Prior to conducting large randomized controlled trials, an in-depth understanding of the changes induced by rIL-7 or nivolumab (and their differences) in patients with sepsis is imperative. We performed a prospective observational cohort study including patients admitted to the intensive care unit with sepsis and characterized their T lymphocyte phenotype using flow cytometry. The ability of T lymphocytes to respond to a stimulus (using anti-CD3/CD28 beads) and the effect of rIL-7 or nivolumab on T lymphocyte immunophenotype ex vivo was assessed. In a cohort of 55 patients, CD4+ and CD8+ T lymphocyte PD-1 was higher and IL-7R lower compared with healthy volunteers. In a subset of 24 intensive care unit patients in whom in-depth immunophenotype was characterized, ex vivo response of lymphocytes to anti-CD3/CD28 beads was reduced compared with healthy volunteers, simultaneously inducing features consistent with immune activation and immunosuppression. rIL-7 was associated with a greater spectrum of changes compared with nivolumab. The response to rIL-7 and nivolumab was influenced by anti-CD3/CD28 bead costimulation. rIL-7 and nivolumab elicited distinct T lymphocyte responses ex vivo, and the changes were influenced by T lymphocyte activation. It needs to be determined if similar changes occur in vivo, which may influence the choice of immunomodulatory therapy in sepsis.
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.
