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Effective eradication of leukemic stem cells (LSCs) remains the greatest challenge in treating acute myeloid leukemia (AML). The immune receptor LAIR-1 has been shown to regulate LSC survival; however, the therapeutic potential of this pathway remains unexplored. We developed a therapeutic LAIR-1 agonist antibody, NC525, that induced cell death of LSCs, but not healthy hematopoietic stem cells in vitro, and killed LSCs and AML blasts in both cell- and patient-derived xenograft models. We showed that LAIR-1 agonism drives a unique apoptotic signaling program in leukemic cells that was enhanced in the presence of collagen. NC525 also significantly improved the activity of azacitidine and venetoclax to establish LAIR-1 targeting as a therapeutic strategy for AML that may synergize with standard-of-care therapies.

The pathophysiology of sickle cell disease (SCD) is driven by chronic inflammation fueled by damage associated molecular patterns (DAMPs). We show that elevated cell-free DNA (cfDNA) in patients with SCD is not just a prognostic biomarker, it also contributes to the pathological inflammation. Within the elevated cfDNA, patients with SCD had a significantly higher ratio of cell-free mitochondrial DNA (cf-mtDNA)/cell-free nuclear DNA compared with healthy controls. Additionally, mitochondrial DNA in patient samples showed significantly disproportionately increased hypomethylation compared with healthy controls, and it was increased further in crises compared with steady-state. Using flow cytometry, structured illumination microscopy, and electron microscopy, we showed that circulating SCD red blood cells abnormally retained their mitochondria and, thus, are likely to be the source of the elevated cf-mtDNA in patients with SCD. Patient plasma containing high levels of cf-mtDNA triggered the formation of neutrophil extracellular traps (NETs) that was substantially reduced by inhibition of TANK-binding kinase 1, implicating activation of the cGAS-STING pathway. cf-mtDNA is an erythrocytic DAMP, highlighting an underappreciated role for mitochondria in sickle pathology. These trials were registered at www.clinicaltrials.gov as #NCT00081523, #NCT03049475, and #NCT00047996.

The formation of neutrophil extracellular traps (NETs) is thought to play a critical role in infections and propagating sterile inflammation. Histone citrullination is an essential and early step in NETs formation, detectable prior to the formation of the hallmark extracellular DNA-scaffolded strands. In addition to the classical microscopy method, new technologies are being developed for studies of NETs and their detection, both for research and clinical purposes. Classical microscopy studies of NETs are subjective, low throughput and semi-quantitative, and limited in their ability to capture the early steps. We have developed this novel Imaging Flow Cytometry (IFC) method that specifically identifies and quantifies citrullination of histone H4 as a NETs marker and its relationship with other alterations at nuclear and cellular level. These include nuclear decondensation and super-condensation, multi-lobulated nuclei versus 1-lobe nuclei and cell membrane damage. NETs markers can be quantified following variable periods of treatment with NETs inducers, prior to the formation of the specific extracellular DNA-scaffolded strands. Because these high throughput image-based cell analysis features can be performed with statistical rigor, this protocol is suited for both experimental and clinical applications as well as clinical evaluations of NETosis as a biomarker., Competing Interests: Competing interestsThe authors declare no competing financial or non-financial interests., (Copyright © 2021 The Authors; exclusive licensee Bio-protocol LLC.)

Neutrophil extracellular traps (NETs) formation has been implicated in an increasing number of infectious and non-infectious pathologies. NETosis is a tightly regulated process; the end-stage and read-out is the formation of DNA strands extruded from the nuclei, and traditionally assessed by fluorescence microscopy. Since NETosis has emerged as a possible biomarker of the inflammatory process, there is a need for less time-consuming, consistent, and quantitative approaches to improve its application in clinical assessment of pro-inflammatory conditions. Imaging Flow Cytometry (IFC) combines features of conventional flow cytometry with qualitative power of fluorescence microscopy and has an added advantage of the capability of assessing the early processes leading up to extrusion of the DNA-scaffolded strands. We explored the optimal imaging-based tools that can be used to measure citrullination of H4 in early NETosis. IFC identified and quantified histone 4 citrullination (H4cit3) induced with several known NETosis stimuli (Ionophore, PMA, LPS, Hemin, and IL-8) following treatment periods ranging from 2 to 60 min. Its relationship with other alterations at nuclear and cellular level, such as nuclear decondensation and super-condensation, multi-lobulated nuclei vs. 1-lobe nuclei and cell membrane damage, were also quantified. We show that the early progress of the H4cit3 response in NETosis depends on the stimulus. Our method identifies fast (Ionophore and Hemin), intermediate and slow (PMA) inducers and shows that H4cit3 appears to have a limited contribution to both early LPS- and IL-8-induced NETosis. While this method is rapid and of a higher throughput compared to fluorescence microscopy, detection and quantification is limited to H4cit3-mediated nuclear events and is likely to be stimulus- and signaling pathway dependent., (Copyright © 2020 Barbu, Dominical, Mendelsohn and Thein.)

Recurring episodes of acute pain, also referred to as vaso-occlusive crises (VOC), are characteristic of sickle cell disease (SCD), during which pro-inflammatory cytokines, chemokines, adhesion markers and white cell count, some already elevated at steady state, increase further. Hydroxyurea (HU) is licensed by the FDA for reducing frequency of VOCs in SCD; increased fetal hemoglobin (HbF) together with reduction of the neutrophil count and circulating inflammatory markers, contribute to its clinical efficacy. Here, using paired plasma samples from HbSS patients (in steady-state and VOC) we determined that despite HU treatment, the SCD environment remained highly inflammatory and particularly at VOC, triggered neutrophil activity. While neutrophil extracellular traps (NETs) induction by the steady state plasmas were comparable to that of plasma from healthy donors, the NETs response triggered by crisis plasmas was significantly increased over that of the steady state (P = 0.0124*). Levels of IL-6 and IL-1α, IL-1ra/IL1F3 and adhesion molecule P-selectin were significantly increased in the VOC plasma when compared with steady state plasma. Higher levels of IL-6 and IL-1ra were also found in the crises samples that yielded an increased NETs response suggesting that increased NETs production associated with increased levels of the inflammatory products of the IL-6 family and regulators of IL-1 family of cytokines during sickle VOCs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Neutrophilia is a feature of sickle cell disease (SCD) that has been consistently correlated with clinical severity and has been shown to remain highly activated even at steady state. In addition to induction of fetal hemoglobin (HbF), hydroxyurea (HU) leads to reduction in neutrophil count and their adhesion properties, which contributes to the clinical efficacy of HU in SCD. Although HU reduces the frequency and severity of acute vaso-occlusive crises (VOCs) and chest syndrome, HU therapy does not abolish these acute clinical events. In this study we investigated whether neutrophils in SCD patients whilst on HU therapy retained features of detrimental pro-inflammatory activity. Freshly isolated neutrophils from SCD patients on HU therapy at steady state and from ethnic-matched healthy controls were evaluated ex vivo for their degranulation response and production of neutrophil extracellular traps (NETs). Unstimulated SCD patient neutrophils already produced NETs within 30 minutes, compared to none for healthy neutrophils, and by 4 hours, these neutrophils produced significantly more NETs than the control neutrophils (P = 0.0079**). Higher numbers of neutrophils from SCD patients also showed higher degree of degranulation-related intracellular features compared to healthy neutrophils, including rough-textured cellular membranes (P = 0.03*), double-positivity for F-Actin and CD63 (P = 0.02*) and re-located CD63 within cytoplasm more efficiently than their healthy counterparts (P = 0.02*). The neutrophils from SCD donors released more myeloperoxidase (P = 0.02*) in the absence of any trigger. Our data showed that neutrophils from patients with SCD at steady state remained active during hydroxyurea treatment and are likely to be able to contribute to the SCD pro-inflammatory environment., Competing Interests: The authors have declared that no competing interests exist.

Objective: We have previously established that the gene for neutrophil cytosolic factor 2 (NCF-2) predisposes to lupus, and we have identified lupus patients with point mutations that are predicted to cause reduced NADPH oxidase activity. We undertook this study to investigate the relationship between reduced leukocyte NADPH oxidase activity and immune dysregulation associated with systemic lupus erythematosus (SLE)., Methods: We generated NCF-2-null mice, in which NADPH oxidase activity is absent, on the nonautoimmune C57BL/6 (B6) mouse background and on the NZM 2328 mouse background, a polygenic model in which mice spontaneously develop lupus. Clinical disease, serology, and immunopathology were evaluated., Results: NCF-2-null mice on the B6 background were susceptible to Aspergillus fumigatus pneumonia characteristic of chronic granulomatous disease, but did not develop systemic lupus disease. In contrast, NCF-2-null and even NCF-2-haploinsufficient mice on the NZM 2328 background developed accelerated full-blown lupus with significantly accelerated lupus kidney disease. This was characterized by more rapid development of hyperactive B cell and T cell immune compartments, increased expression of type I interferon-responsive genes, and generation of neutrophil extracellular traps, which were observed even in the absence of NADPH oxidase activity., Conclusion: Just as patients with chronic granulomatous disease who lack NADPH oxidase rarely develop SLE, NCF-2-null mice on a nonautoimmune background were susceptible to a chronic granulomatous disease-like opportunistic infection but did not develop lupus. In contrast, on a lupus-prone background, even haploinsufficiency of NCF-2 accelerated the development of full-blown lupus disease. This establishes an interaction between reduced oxidase activity and other lupus-predisposing genes, paralleling human SLE-associated variants predicted to have only reduced NADPH oxidase activity., (© 2017, American College of Rheumatology.)

Mutations in the leukocyte NADPH oxidase that abrogate superoxide production result in chronic granulomatous disease (CGD), an inherited immunodeficiency associated with recurrent infections and inflammatory complications. The cytosolic regulatory subunit p40 phox plays a specialized role in stimulating NADPH oxidase activity on intracellular membranes via its phosphatidylinositol 3-phosphate [PI(3)P]-binding domain, as revealed by studies largely focused on neutrophils. Whether PI(3)P-p40 phox -regulated superoxide production contributes to regulating inflammatory responses is not well understood. Here, we report that mice expressing p40 phox R58A, which lacks PI(3)P binding, had impaired macrophage NADPH oxidase activity and increased sterile inflammation. p40 phoxR58A/R58A macrophages exhibited diminished phagosome reactive oxygen species (ROS) in response to certain particulate and soluble ligands, including IgG-opsonized particles and a TLR2 agonist, along with unexpected defects in plasma membrane oxidase activity. Compared with wild-type (WT) mice, p40 phoxR58A/R58A mice had elevated numbers of newly recruited neutrophils and monocytes in peritoneal inflammation elicited by zymosan, monosodium urate (MSU) crystals, or sodium periodate. At later time points, higher numbers of inflammatory macrophages in p40 phoxR58A/R58A mice were consistent with delayed resolution. Our studies demonstrate a critical role of PI(3)P-p40 phox binding for optimal activation of the NADPH oxidase in macrophages. Furthermore, selective loss of PI(3)P-regulated NADPH oxidase activity was sufficient to enhance significantly responses to inflammation and delay resolution., (© Society for Leukocyte Biology.)

Mast cell activation results in the release of stored and newly synthesized inflammatory mediators. We found that Zeb2 (also named Sip1, Zfhx1b), a zinc finger transcription factor, regulates both early and late mast cell responses. Transfection with small interfering RNA (siRNA) reduced Zeb2 expression and resulted in decreased FcεRI-mediated degranulation, with a parallel reduction in receptor-induced activation of NFAT and NF-κB transcription factors, but an enhanced response to the LPS-mediated activation of NF-κB. There was variable and less of a decrease in the Ag-mediated release of the cytokines TNF-α, IL-13, and CCL-4. This suggests that low Zeb2 expression differentially regulates signaling pathways in mast cells. Multiple phosphorylation events were impaired that affected molecules both at early and late events in the signaling pathway. The Zeb2 siRNA-treated mast cells had altered cell cycle progression, as well as decreased expression of several molecules including cell surface FcεRI and its β subunit, Gab2, phospholipase-Cγ1, and phospholipase-Cγ2, all of which are required for receptor-induced signal transduction. The results indicate that the transcription factor Zeb2 controls the expression of molecules thereby regulating signaling in mast cells.

Activation of the high affinity IgE-binding receptor (FcεRI) results in the tyrosine phosphorylation of two conserved tyrosines located close to the COOH terminus of the protein-tyrosine kinase Syk. Synthetic peptides representing the last 10 amino acids of the tail of Syk with these two tyrosines either nonphosphorylated or phosphorylated were used to precipitate proteins from mast cell lysates. Proteins specifically precipitated by the phosphorylated peptide were identified by mass spectrometry. These included the adaptor proteins SLP-76, Nck-1, Grb2, and Grb2-related adaptor downstream of Shc (GADS) and the protein phosphatases SHIP-1 and TULA-2 (also known as UBASH3B or STS-1). The presence of these in the precipitates was further confirmed by immunoblotting. Using the peptides as probes in far Western blots showed direct binding of the phosphorylated peptide to Nck-1 and SHIP-1. Immunoprecipitations suggested that there were complexes of these proteins associated with Syk especially after receptor activation; in these complexes are Nck, SHIP-1, SLP-76, Grb2, and TULA-2 (UBASH3B or STS-1). The decreased expression of TULA-2 by treatment of mast cells with siRNA increased the FcεRI-induced tyrosine phosphorylation of the activation loop tyrosines of Syk and the phosphorylation of phospholipase C-γ2. There was parallel enhancement of the receptor-induced degranulation and activation of nuclear factor for T cells or nuclear factor κB, indicating that TULA-2, like SHIP-1, functions as a negative regulator of FcεRI signaling in mast cells. Therefore, once phosphorylated, the terminal tyrosines of Syk bind complexes of proteins that are positive and negative regulators of signaling in mast cells.

The aggregation by antigen of the IgE bound to its high affinity receptor on mast cells initiates a complex series of biochemical events that result in the release of inflammatory mediators. The essential role of the protein tyrosine kinase Syk has been appreciated for some time, and newer results have defined the mechanism of its activation. The use of siRNA has defined the relative contribution of Syk, Fyn and Gab2 to signaling and has made possible a screening study to identify previously unrecognized molecules that are involved in these pathways., (Published by Elsevier B.V.)

Mast cells play pivotal roles in the initiation of the allergic response. To gain an understanding of the functions played by phosphatases in IgE-mediated mast cell activation, a small interfering RNA (siRNA) library that targets all mouse phosphatase genes was screened in a mouse mast cell line, MMC-1. Of 198 targets, 10 enhanced and 7 inhibited FcepsilonRI-induced degranulation. For seven of the strongest hits, four different siRNAs per target were tested, and at least two out of the four single siRNA per target had similar effects as the pool suggesting that these were true hits. Bone marrow-derived mast cells from normal mice further validated these results for six definite positive targets. The mechanism of the reduced mast cell degranulation due to calcineurin B deficiency was investigated. Calcineurin B deficiency reduced the phosphorylation of MAPKs and the phosphorylation of protein kinase D/protein kinase Cmu and protein kinase Cdelta, which are involved in FcepsilonRI signaling. The screen, therefore, has identified several new molecules that are critical for FcepsilonRI-induced degranulation. Regulating the function of these proteins may be potential targets for the treatment of allergic inflammation. The result also indicates that the system used is efficient for searching molecules implicated in complex receptor-induced signaling.

Several studies with mast cells from knock-out mice have suggested that the tyrosine kinase Fyn and its downstream substrate Gab2 may play a role in high affinity IgE receptor (FcepsilonRI)-mediated mast cell activation. To better understand the role of these two molecules and of Syk, we transiently transfected mast cells with small interference RNA (siRNA) targeted to Fyn, Gab2, or Syk to specifically decrease their expression. The siRNA suppression of Gab2 but not Fyn reduced activation of the phosphoinositide-3-kinase (PI3K) pathway as demonstrated by the change in phosphorylation of Akt; this indicates that Gab2 but not Fyn regulates this pathway. The decreased expression of Gab2 and Fyn had minor effects on degranulation. There were also some minor changes in activation of the NFAT or NFkappaB transcription factors in cells with reduced expression of Fyn or Gab2. Decreased Gab2 but not Fyn reduced the FcepsilonRI-induced activation of the Erk, Jnk, and p38 MAP kinases and the release of TNF-alpha. In contrast, decreased expression of Syk dramatically reduced FcepsilonRI-induced degranulation, activation of NFAT and NFkappaB. Therefore, the reduction in expression of these proteins in mast cells indicates that Syk is the major regulator of FcepsilonRI-mediated reactions, whereas Fyn has minor if any effects and Gab2 regulates primarily late events including MAP kinase activation and release of cytokines.

While the current understanding of the stimulus-response coupling networks triggered by the multi-chain immune-recognition receptors (MIRRs) has markedly advanced, knowledge of its control mechanisms is only emerging. Regulation of the secretory response of mast cells to the stimulus provided by the type I Fcepsilon receptor (FcepsilonRI) is our topic of interest. Several mast cell membrane receptors capable of inhibiting both immediate and late responses have so far been identified. However, their ligands and mechanism(s) of operation are only partly known. Moreover, desensitization of mast cells' response to the FcepsilonRI, a well-known and widespread control process of many neural or hormone receptors, is hardly understood in this case. In this brief report we describe results of recent experiments in which we studied both of these aspects of mast cells' response to the FcepsilonRI stimulus by an inhibitory receptor MAFA, as well as those where we have established that these cells are susceptible to physiological modes of FcepsilonRI desensitization caused by prolonged exposure to sub-threshold concentrations of FcepsilonRI clustering agents.