Apoptotic Neutrophils Research Articles

MerTK-dependent efferocytosis by monocytic-MDSCs mediates resolution of ischemia/reperfusion injury after lung transplant.

Lung transplantation (LTx) outcomes are impeded by ischemia/reperfusion injury (IRI) and subsequent chronic lung allograft dysfunction (CLAD). We examined the undefined role of receptor Mer tyrosine kinase (MerTK) on monocytic myeloid-derived suppressor cells (M-MDSCs) in efferocytosis to facilitate resolution of lung IRI. Single-cell RNA sequencing of lung tissue and bronchoalveolar lavage (BAL) from patients after LTx were analyzed. Murine lung hilar ligation and allogeneic orthotopic LTx models of IRI were used with BALB/c (WT), Cebpb-/- (MDSC-deficient), Mertk-/-, or MerTK-cleavage-resistant mice. A significant downregulation in MerTK-related efferocytosis genes in M-MDSC populations of patients with CLAD was observed compared with healthy individuals. In the murine IRI model, a significant increase in M-MDSCs, MerTK expression, and efferocytosis and attenuation of lung dysfunction was observed in WT mice during injury resolution that was absent in Cebpb-/- and Mertk-/- mice. Adoptive transfer of M-MDSCs in Cebpb-/- mice significantly attenuated lung dysfunction and inflammation. Additionally, in a murine orthotopic LTx model, increases in M-MDSCs were associated with resolution of lung IRI in the transplant recipients. In vitro studies demonstrated the ability of M-MDSCs to efferocytose apoptotic neutrophils in a MerTK-dependent manner. Our results suggest that MerTK-dependent efferocytosis by M-MDSCs can substantially contribute to the resolution of post-LTx IRI.

Hybrid biomaterial hydrogel loading iRGD&PS double modified lipid nanoparticles ameliorates diabetic wound healing through promoting efferocytosis and glycolysis-related macrophage reprogramming

Efferocytosis is a critical process whereby macrophages residing in the wound area play a key role in the efficient clearing and degradation of apoptotic neutrophils. This process is followed by a phenotypic transition toward an anti-inflammatory state, essential for inflammation resolution and tissue repair. The cystine/glutamate antiporter SLC7A11 has recently been identified as an inhibitor of efferocytosis, and its blockade has been found to enhance wound healing. In this study, we demonstrated that tiliroside, a plant-derived glycoside containing flavones, binds directly to SLC7A11 and pyruvate kinase isozyme M2 (PKM2). This was established by molecular docking predictions and activity-based protein profiling (ABPP). Cytological experiments revealed that tiliroside promoted the process of efferocytosis and led to glycolysis-related macrophage reprogramming. To facilitate targeted drug delivery to macrophages at diabetic wound sites, we designed a novel hybrid biomaterial. This hybrid biomaterial, prepared as Gel@Til iRGD&PS@PLGA NPs is manufactured by loading tiliroside (Til)-conjugated iRGD&PS double modified lipid nanoparticles (iRGD&PS@PLGA NPs) into a pH-responsive hydrogel matrix. The administration of Gel@Til iRGD&PS@PLGA NPs in diabetic cutaneous wound models has been shown to significantly promote tissue regeneration through the promotion of efferocytosis and glycolysis-related macrophage reprogramming. Therefore, this study introduces a novel approach to diabetic wound management by leveraging the promotion of efferocytosis.

Dietary and water restriction leads to increased susceptibility to antimicrobial resistant pathogens.

Dehydration and malnutrition are common and often underdiagnosed in hospital settings. Multidrug-resistant bacterial infections result in more than 35,000 deaths a year in nosocomial patients. The effect of temporal dietary and water restriction (DWR) on susceptibility to multidrug-resistant pathogens is unknown. We report that DWR markedly increased susceptibility to systemic infection by ESKAPE pathogens. Using a murine bloodstream model of methicillin-resistant Staphylococcus aureus infection, we show that DWR leads to significantly increased mortality and morbidity. DWR causes increased bacterial burden, severe pathology, and increased numbers of phagocytes in the kidney. DWR appears to alter the functionality of these phagocytes and is therefore unable to control infection. Mechanistically, we show that DWR impairs the ability of macrophages to phagocytose multiple bacterial pathogens and efferocytose apoptotic neutrophils. Together, this work highlights the crucial impact that diet and hydration play in protecting against infection.

Tagetes erecta L.: A traditional medicine effective in inflammatory process treatment

Ethnopharmacological relevanceTagetes erecta L. (Asteraceae), popularly known as Aztec Marigold, is used in folk medicine to treat several ailments including inflammatory processes. Despite its historical use, the specific mechanisms through which it may modulate inflammation, particularly its effects on neutrophils and macrophages activation, have not yet been completely investigated. Aim of the studyThis study aimed to elucidate the anti-inflammatory mechanism of the hydroalcoholic extract obtained from T. erecta flowers, focusing on its role in the regulation of neutrophil and macrophage functions. Material and methodsThe production of TNF, IL-6, CXCL-1, IL-1β, IL-10 (ELISA) and NO (Griess reaction), adhesion molecule expression (CD62L, CD49d and CD18, flow cytometry), and chemotaxis were analyzed in vitro using oyster glycogen-recruited peritoneal neutrophils or macrophages (RAW 264.7) stimulated with lipopolysaccharide (LPS) and treated with the extract (1, 10 or 100 μg/mL). The resolution of inflammation was accessed by efferocytosis assay. The in vivo anti-inflammatory activity was investigated using carrageenan-induced inflammation in the subcutaneous tissue of male Swiss mice orally treated with the T. erecta extract (30, 100 or 300 mg/kg). The leukocyte influx (optical microscopy), secretion of chemical mediators (TNF, IL-6 and IL-1β, ELISA) and protein exudation (Bradford reaction) were quantified in the inflamed exudate. ResultsIn vitro studies demonstrated that the extract inhibited neutrophil chemotaxis and reduced the production and/or release of cytokines (TNF, IL-1β, CXCL1, and IL-6) as well as nitric oxide (NO) by neutrophils and macrophages when stimulated with LPS. Neutrophils treated with LPS and incubated with the extract showed an increase in CD62L expression, which leads to the impairment of neutrophil adhesion. The extract also enhanced efferocytosis of apoptotic neutrophils by macrophages, which was accompanied by increased IL-10 secretion and decreased TNF levels. In vivo studies yielded similar results, showing reduction in neutrophil migration, protein exudation, and cytokine release (TNF, IL-6, and IL-1β). ConclusionsTogether, the data herein obtained shows that T. erecta flower extract has anti-inflammatory effects by regulating inflammatory mediators, limiting neutrophil migration, and promoting efferocytosis. The in vivo results suggest that an herbal medicine made with T. erecta could represent an interesting pharmacological tool for the treatment of acute inflammatory condition.

Cholinergic macrophages promote the resolution of peritoneal inflammation

The non-neural cholinergic system plays a critical role in regulating immune equilibrium and tissue homeostasis. While the expression of choline acetyltransferase (ChAT), the enzyme catalyzing acetylcholine biosynthesis, has been well documented in lymphocytes, its role in the myeloid compartment is less understood. Here, we identify a significant population of macrophages (Mϕs) expressing ChAT and synthesizing acetylcholine in the resolution phase of acute peritonitis. Using Chat-GFP reporter mice, we observed marked upregulation of ChAT in monocyte-derived small peritoneal Mϕs (SmPMs) in response to Toll-like receptor agonists and bacterial infections. These SmPMs, phenotypically and transcriptionally distinct from tissue-resident large peritoneal macrophages, up-regulated ChAT expression through a MyD88-dependent pathway involving MAPK signaling. Notably, this process was attenuated by the TRIF-dependent TLR signaling pathway, and our tests with a range of neurotransmitters and cytokines failed to induce a similar response. Functionally, Chat deficiency in Mϕs led to significantly decreased peritoneal acetylcholine levels, reduced efferocytosis of apoptotic neutrophils, and a delayed resolution of peritonitis, which were reversible with exogenous ACh supplementation. Intriguingly, despite B lymphocytes being a notable ChAT-expressing population within the peritoneal cavity, Chat deletion in B cells did not significantly alter the resolution process. Collectively, these findings underscore the crucial role of Mϕ-derived acetylcholine in the resolution of inflammation and highlight the importance of the non-neuronal cholinergic system in immune regulation.

Activating Macrophage Continual Efferocytosis via Microenvironment Biomimetic Short Fibers for Reversing Inflammation in Bone Repair.

Efferocytosis-mediated inflammatory reversal plays a crucial role in bone repairing process. However, in refractory bone defects, the macrophage continual efferocytosis may be suppressed due to the disrupted microenvironment homeostasis, particularly the loss of apoptotic signals and overactivation of intracellular oxidative stress. In this study, a polydopamine-coated short fiber matrix containing biomimetic "apoptotic signals" to reconstruct the microenvironment and reactivate macrophage continual efferocytosis for inflammatory reversal and bone defect repair is presented. The "apoptotic signals" (AM/CeO2) are prepared using CeO2 nanoenzymes with apoptotic neutrophil membrane coating for macrophage recognition and oxidative stress regulation. Additionally, a short fiber "biomimetic matrix" is utilized for loading AM/CeO2 signals via abundant adhesion sites involving π-π stacking and hydrogen bonding interactions. Ultimately, the implantable apoptosis-mimetic nanoenzyme/short-fiber matrixes (PFS@AM/CeO2), integrating apoptotic signals and biomimetic matrixes, are constructed to facilitate inflammatory reversal and reestablish the pro-efferocytosis microenvironment. In vitro and in vivo data indicate that the microenvironment biomimetic short fibers can activate macrophage continual efferocytosis, leading to the suppression of overactivated inflammation. The enhanced repair of rat femoral defect further demonstrates the osteogenic potential of the pro-efferocytosis strategy. It is believed that the regulation of macrophage efferocytosis through microenvironment biomimetic materials can provide a new perspective for tissue repair.

Carbon monoxide-induced autophagy enhances human mesenchymal stromal cell function via paracrine actions in murine polymicrobial sepsis

Sepsis is a life-threatening process due to organ dysfunction resulting from severe infections. Mesenchymal stromal cells (MSCs) are being investigated as therapy for sepsis, along with conditioning regimens to improve their function. Carbon monoxide (CO) gas, which is cytoprotective at low doses, induces autophagy and is a mediator of inflammation. We evaluated CO-induced autophagy in human MSCs (hMSCs), and its impact on cell function in murine cecal ligation and puncture. Conditioning of hMSCs with CO exvivo resulted in enhanced survival and bacterial clearance invivo, and neutrophil phagocytosis of bacteria invitro. Decreased neutrophil infiltration and less parenchymal cell death in organs were associated with increased macrophage efferocytosis of apoptotic neutrophils, promoting resolution of inflammation. These CO effects were lost when the cells were exposed to autophagy inhibition prior to gas exposure. When assessing paracrine actions of CO-induced autophagy, extracellular vesicles (EVs) were predominantly responsible. CO had no effect on EV production, but altered their miRNA cargo. Increased expression of miR-145-3p and miR-193a-3p by CO was blunted with disruption of autophagy, and inhibitors of these miRNAs led to a loss of neutrophil phagocytosis and macrophage efferocytosis. Collectively, CO-induced autophagy enhanced hMSC function during sepsis via paracrine actions of MSC-derived EVs.

P120 catenin enhances macrophage efferocytosis and facilitates resolution of lung inflammatory injury

Abstract Efficient clearance of apoptotic neutrophils (PMNs) (efferocytosis) after sepsis plays a crucial role in facilitating tissue repair, resolving inflammation, and maintaining immune system balance. Here, we identified p120 catenin (p120) as a pivotal regulator in efferocytosis and subsequent recovery from inflammatory lung injury. We found that specific macrophage p120 deletion had a profound delay in the resolution of PMN infiltration, extracellular protein accumulation, and lung edema and injury following LPS challenge. p120 deletion increased the levels of TNF-α and IL-6, and reduced the levels of TGF-β1 and IL-10. Depletion of p120 also led to a marked decrease in the phagocytosis of apoptotic PMNs by macrophages. The number of p120-depleted alveolar macrophages containing apoptotic PMNs in bronchoalveolar lavage fluid was significantly lower. p120 deficiency caused a reduction in the expression of key efferocytosis-related molecules, namely AXL and CD36. p120-depleted macrophages exhibited a shift in polarization towards the M1 phenotype. Apoptotic cells triggered the association and co-localization of p120 and PPAR, and p120 deletion decreased the activity of PPARgamma in response to apoptotic PMNs. Inhibition of PPAR abolished p120-mediated efferocytosis and the resolution of lung inflammation. These findings underscore the critical role of p120 in facilitating the resolution of lung inflammation through modulation of macrophage polarization and PPAR signaling pathway.

Building erudition in the wound healing process: an inflammation model analysis

The main four stages of wound healing are hemostasis, inflammation, proliferation, and remodelling. In the inflammation stage, the body rids of pathogens and debris, and prepares for tissue regrowth. An ordinary differential equation model of the inflammation is constructed that expands on previous models and incorporates dynamics reported in the updated literature. The interaction between debris and apoptotic neutrophils, along with classical and alternative macrophages are included. In all, the model incorporates pathogens, debris, apoptotic neutrophils, neutrophil, M1 macrophages, and M2 macrophages. Parameter values are analysed based on 1. experimental data and 2. reported ranges and expected peak times of white blood cells. Next, global sensitivity analyses which is able to test change in parameter values in tandem is conducted to interpret the overall influence each parameter has on white blood cell, debris, and pathogen output.

Apoptotic cell identity induces distinct functional responses to IL-4 in efferocytic macrophages.

Macrophages are functionally heterogeneous cells essential for apoptotic cell clearance. Apoptotic cells are defined by homogeneous characteristics, ignoring their original cell lineage identity. We found that in an interleukin-4 (IL-4)-enriched environment, the sensing of apoptotic neutrophils by macrophages triggered their tissue remodeling signature. Engulfment of apoptotic hepatocytes promoted a tolerogenic phenotype, whereas phagocytosis of T cells had little effect on IL-4-induced gene expression. In a mouse model of parasite-induced pathology, the transfer of macrophages conditioned with IL-4 and apoptotic neutrophils promoted parasitic egg clearance. Knockout of phagocytic receptors required for the uptake of apoptotic neutrophils and partially T cells, but not hepatocytes, exacerbated helminth infection. These findings suggest that the identity of apoptotic cells may contribute to the development of distinct IL-4-driven immune programs in macrophages.

In situ neutrophil apoptosis and macrophage efferocytosis mediated by Glycyrrhiza protein nanoparticles for acute inflammation therapy

In the progression of acute inflammation, the activation and recruitment of macrophages and neutrophils are mutually reinforcing, leading to amplified inflammatory response and severe tissue damage. Therefore, to regulate the axis of neutrophils and macrophages is essential to avoid tissue damage induced from acute inflammatory. Apoptotic neutrophils can regulate the anti-inflammatory activity of macrophages through the efferocytosis. The strategy of in situ targeting and inducing neutrophil apoptosis has the potential to modulate macrophage activity and transfer anti-inflammatory drugs. Herein, a natural glycyrrhiza protein nanoparticle loaded with dexamethasone (Dex@GNPs) was constructed, which could simultaneously regulate neutrophil and macrophage function during acute inflammation treatment by combining in situ neutrophil apoptosis and macrophage efferocytosis. Dex@GNPs can be rapidly and selectively internalized by neutrophils and subsequently induce neutrophils apoptosis through a ROS-dependent mechanism. The efferocytosis of apoptotic neutrophils not only promoted the polarization of macrophages into anti-inflammatory state, but also facilitated the transfer of Dex@GNPs to macrophages. This enabled dexamethasone to further modulate macrophage function. In mouse models of acute respiratory distress syndrome and sepsis, Dex@GNPs significantly ameliorated the disordered immune microenvironment and alleviated tissue injury. This study presents a novel strategy for drug delivery and inflammation regulation to effectively treat acute inflammatory diseases.

Single-cell transcriptomics identifies the differentiation trajectory from inflammatory monocytes to pro-resolving macrophages in a mouse skin allergy model

Both monocytes and macrophages are heterogeneous populations. It was traditionally understood that Ly6Chi classical (inflammatory) monocytes differentiate into pro-inflammatory Ly6Chi macrophages. Accumulating evidence has suggested that Ly6Chi classical monocytes can also differentiate into Ly6Clo pro-resolving macrophages under certain conditions, while their differentiation trajectory remains to be fully elucidated. The present study with scRNA-seq and flow cytometric analyses reveals that Ly6ChiPD-L2lo classical monocytes recruited to the allergic skin lesion sequentially differentiate into Ly6CloPD-L2hi pro-resolving macrophages, via intermediate Ly6ChiPD-L2hi macrophages but not Ly6Clo non-classical monocytes, in an IL-4 receptor-dependent manner. Along the differentiation, classical monocyte-derived macrophages display anti-inflammatory signatures followed by metabolic rewiring concordant with their ability to phagocytose apoptotic neutrophils and allergens, therefore contributing to the resolution of inflammation. The failure in the generation of these pro-resolving macrophages drives the IL-1α-mediated cycle of inflammation with abscess-like accumulation of necrotic neutrophils. Thus, we clarify the stepwise differentiation trajectory from Ly6Chi classical monocytes toward Ly6Clo pro-resolving macrophages that restrain neutrophilic aggravation of skin allergic inflammation.

B cell subsets contribute to myocardial protection by inducing neutrophil apoptosis after ischemia and reperfusion.

A robust, sterile inflammation underlies myocardial ischemia and reperfusion injury (MIRI). Several subsets of B cells possess the immunoregulatory capacity that limits tissue damage, yet the role of B cells in MIRI remains elusive. Here, we sought to elucidate the contribution of B cells to MIRI by transient ligation of the left anterior descending coronary artery in B cell-depleted or -deficient mice. Following ischemia and reperfusion (I/R), regulatory B cells are rapidly recruited to the heart. B cell-depleted or -deficient mice exhibited exacerbated tissue damage, adverse cardiac remodeling, and an augmented inflammatory response after I/R. Rescue and chimeric experiments indicated that the cardioprotective effect of B cells was not solely dependent on IL-10. Coculture experiments demonstrated that B cells induced neutrophil apoptosis through contact-dependent interactions, subsequently promoting reparative macrophage polarization by facilitating the phagocytosis of neutrophils by macrophages. The in vivo cardioprotective effect of B cells was undetectable in the absence of neutrophils after I/R. Mechanistically, ligand-receptor imputation identified FCER2A as a potential mediator of interactions between B cells and neutrophils. Blocking FCER2A on B cells resulted in a reduction in the percentage of apoptotic neutrophils, contributing to the deterioration of cardiac remodeling. Our findings unveil a potential cardioprotective role of B cells in MIRI through mechanisms involving FCER2A, neutrophils, and macrophages.

Tissue-Resident Alveolar Macrophages Reduce Ozone-induced Inflammation via MerTK-mediated Efferocytosis.

Lung inflammation, caused by acute exposure to ozone (O3), one of the six criteria air pollutants, is a significant source of morbidity in susceptible individuals. Alveolar macrophages (AMØs) are the most abundant immune cells in the normal lung, and their number increases after O3 exposure. However, the role of AMØs in promoting or limiting O3-induced lung inflammation has not been clearly defined. In this study, we used a mouse model of acute O3 exposure, lineage tracing, genetic knockouts, and data from O3-exposed human volunteers to define the role and ontogeny of AMØs during acute O3 exposure. Lineage-tracing experiments showed that 12, 24, and 72 hours after exposure to O3 (2 ppm) for 3 hours, all AMØs were of tissue-resident origin. Similarly, in humans exposed to filtered air and O3 (200 ppb) for 135 minutes, we did not observe at ∼21 hours postexposure an increase in monocyte-derived AMØs by flow cytometry. Highlighting a role for tissue-resident AMØs, we demonstrate that depletion of tissue-resident AMØs with clodronate-loaded liposomes led to persistence of neutrophils in the alveolar space after O3 exposure, suggesting that impaired neutrophil clearance (i.e., efferocytosis) leads to prolonged lung inflammation. Moreover, depletion of tissue-resident AMØs demonstrated reduced clearance of intratracheally instilled apoptotic Jurkat cells, consistent with reduced efferocytosis. Genetic ablation of MerTK (MER proto-oncogene, tyrosine kinase), a key receptor involved in efferocytosis, also resulted in impaired clearance of apoptotic neutrophils after O3 exposure. Overall, these findings underscore the pivotal role of tissue-resident AMØs in resolving O3-induced inflammation via MerTK-mediated efferocytosis.

Calorie restriction mimetic, resveratrol, attenuates hepatic ischemia and reperfusion injury through enhancing efferocytosis of macrophages via AMPK/STAT3/S1PR1 pathway

Calorie restriction (CR) mimetic, resveratrol (RSV), has the capacity of promoting phagocytosis. However, its role in hepatic ischemia and reperfusion injury (HIRI) remains poorly understood. This study aimed to investigate the effect of RSV on alleviating HIRI and explore the underlying mechanisms. RSV was intraperitoneally injected in mice HIRI model, while RSV was co-incubated with culture medium for 24 h in RAW 264.7 cells and kupffer cells. Macrophage efferocytosis was assessed by immunostaining of PI and F4/80. The clearance of apoptotic neutrophils in the liver was determined by immunostaining of Ly6-G and cleaved-caspase-3. HE staining, Suzuki's score, serum levels of ALT, AST, TNF-α and IL-1β were analyzed to evaluate HIRI. The efferocytosis inhibitor, Cytochalasin D, was utilized to investigate the effect of RSV on HIRI. Western blot was employed to measure the levels of AMPKα, phospho-AMPKα, STAT3, phospho-STAT3 and S1PR1. SiSTAT3 and inhibitors targeting AMPK, STAT3 and S1PR1, respectively, were used to confirm the involvement of AMPK/STAT3/S1PR1 pathway in RSV-mediated efferocytosis and HIRI. RSV facilitated the clearance of apoptotic neutrophils and attenuated HIRI, which was impeded by Cytochalasin D. RSV boosted macrophage efferocytosis by up-regulating the levels of phospho-AMPKα, phospho-STAT3 and S1PR1, which was reversed by AMPK, STAT3 and S1PR1 inhibitors, respectively. Inhibition of STAT3 suppressed RSV-induced clearance of apoptotic neutrophils and exacerbated HIRI. CR mimetic, RSV, alleviates HIRI by promoting macrophages efferocytosis through AMPK/STAT3/S1PR1 pathway, providing valuable insights into the mechanisms underlying the protective effects of CR on attenuating HIRI.

Gasdermin E dictates inflammatory responses by controlling the mode of neutrophil death

Both lytic and apoptotic cell death remove senescent and damaged cells in living organisms. However, they elicit contrasting pro- and anti-inflammatory responses, respectively. The precise cellular mechanism that governs the choice between these two modes of death remains incompletely understood. Here we identify Gasdermin E (GSDME) as a master switch for neutrophil lytic pyroptotic death. The tightly regulated GSDME cleavage and activation in aging neutrophils are mediated by proteinase-3 and caspase-3, leading to pyroptosis. GSDME deficiency does not alter neutrophil overall survival rate; instead, it specifically precludes pyroptosis and skews neutrophil death towards apoptosis, thereby attenuating inflammatory responses due to augmented efferocytosis of apoptotic neutrophils by macrophages. In a clinically relevant acid-aspiration-induced lung injury model, neutrophil-specific deletion of GSDME reduces pulmonary inflammation, facilitates inflammation resolution, and alleviates lung injury. Thus, by controlling the mode of neutrophil death, GSDME dictates host inflammatory outcomes, providing a potential therapeutic target for infectious and inflammatory diseases.

Morphological abnormalities of leukocytes in SARS-CoV-2 infection.

The causative agent of COVID-19 (Coronavirus Disease 2019) is an enveloped RNA (ribonucleic acid) virus of the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus-2) family. The effects of SARS-CoV-2 on the differentiation and maturation of blood cells have been the subject of several studies, we report our experience of an investigation of the morphologic abnormalities of leukocytes observed during COVID-19. This is a prospective study of 5 months, from February 2021 to June 2021. Forty COVID-19 patients and 20 healthy controls were included in this study. We performed complete blood count and peripheral blood smear of all patients and control samples. Leukocytes abnormalities were quantified as a percentage of 100 leukocytes of the same lineage. The morphological abnormalities of the leukocytes found in percentage of patients have been mainly neutrophils bilobed 72,5%, hypogranulation 45%, acquired pseudo Pelger-Huet 35%, vacuolated neutrophils 42,5%, Apoptotic neutrophils 35,5 %, neutrophils with toxic granulations 30%, myelemia 45%, atypical lymphocytes 52,5%, lymphoplasmocytes 60% and vacuolated monocytes 27, 5%. Our study revealed several morphological abnormalities of the different cells of the leukocyte lineage. The presence of toxic granulations in the cytoplasm of the myelocytes was specific to this study.

NET formation is a default epigenetic program controlled by PAD4 in apoptotic neutrophils.

Neutrophil extracellular traps (NETs) not only counteract bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. Mitochondrial- and death receptor-mediated apoptosis promote gasdermin E (GSDME)-dependent calcium mobilization and membrane permeabilization leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit is concentrated at the promoter in bone marrow neutrophils and redistributes in a coordinated process from promoter to intergenic and intronic regions during apoptosis. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils but prolongs early apoptosis-induced cellular changes to the chromatin and cytoplasmic granules. Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end of life for neutrophils.

Gsdme Promotes PAD4 Activation and DNA Externalization from Apoptotic Neutrophils

Introduction Neutrophil extracellular traps (NETs) neutralize bacterial and fungal pathogens but can also promote thrombosis, autoimmunity, and sterile inflammation. The presence of citrullinated histones, generated by the peptidylarginine deiminase 4 (PAD4), is synonymous with NETosis and is considered independent of apoptosis. However, non-apoptotic cell death signaling pathways mediated by MLKL and GSDMD can also engage PAD4 in neutrophils to trigger NET formation. Additionally, while PAD4 is hypothesized to neutralize charge on histones to facilitate chromatin decondensation, a genetic deficiency in Padi4 fails to prevent chromatin decondensation but does prevent NET formation following MLKL activation. Together, these data suggest more complex roles for PAD4 in the extrusion of nuclear DNA. Here we explored the role of GSDME - another pore-forming protein activated by apoptotic caspases - in controlling PAD4 activation, DNA externalization, and NET formation in apoptotic neutrophils. Methods Cell viability was quantified by flow cytometry and live-cell imaging using Hoechst, Cell Tracker Green, Annexin V, and propidium iodide. Histone citrullination was studied at the population level by immunoblot, at the single cell level by lattice SIM super-resolution microscopy, and at the molecular level by an in-situ ChIP-Seq methodology CUT&Tag. Calcium signaling linked to PAD4 activation was monitored by flow cytometry. Ultrastructural changes occurring in the absence of PAD4 and GSDME were investigated by transmission electron microscopy and super-resolution microscopy. Results Intrinsic and extrinsic apoptosis promote GSDME-dependent calcium mobilization and membrane permeabilization, leading to histone H3 citrullination (H3Cit), nuclear DNA extrusion, and cytoplast formation. H3Cit distribution on chromatin is developmentally controlled by PAD4 in neutrophils, with H3Cit found concentrated at promoter regions during neutrophil development. Lattice SIM super-resolution microscopy revealed clustered distribution of H3Cit in the nuclei of neutrophils, reflecting a unique spatial organization and coordination of multiple promoter elements in neutrophils. Following apoptotic stimulation, H3Cit redistributes in a coordinated process from promoter to intergenic and intronic regions. Loss of GSDME prevents nuclear and plasma membrane disruption of apoptotic neutrophils, but also blocks calcium signaling that activates PAD4, ultimately preventing NET formation. During apoptosis in GSDME-deficient neutrophils, early-apoptotic cellular changes to the chromatin and cytoplasmic granules are prolonged, generating highly atypical cellular states that fail to extrude nuclear DNA. Conclusion Apoptotic signaling engages PAD4 in neutrophils, establishing a cellular state that is primed for NETosis, but that occurs only upon membrane disruption by GSDME, thereby redefining the end-of-life for neutrophils.

Isocitrate Dehydrogenase 2 Mutation Allows Myeloid Differentiation but Impairs Bone Marrow Macrophage Polarization and Function Via Metabolic Dysregulation

Isocitrate dehydrogenase 2 (IDH2) mutations, while less common than other mutations associated with myelodysplastic syndromes (MDS), are also found in clonal hematopoiesis and are associated with increased risk of transformation to leukemia. IDH2 mutations lead to metabolic dysfunction and DNA hypermethylation due to the accumulation of D-2-hydroxyglutarate (2HG), an oncometabolite derived from alpha-ketoglutarate (aKG), which disrupts hematopoiesis. However, the mechanisms that induce clonal progression and transformation remain incompletely understood. Furthermore, the intrinsic and extrinsic impact of IDH mutations on mature myeloid cell functions and the bone marrow microenvironment remains to be elucidated. Previous literature suggested that hypermethylation via this mutant leads to incomplete differentiation and failed mature myeloid cell generation. Our lab previously demonstrated that efferocytic defects in bone marrow macrophages can skew hematopoietic stem and progenitor cells via increased inflammatory mediators. Based on these data, we hypothesized that IDH2 mutations prevent complete polarization down pro-efferocytic phenotypes in macrophages due to blocking of metabolic pathways required for mature macrophage function. Congenic CD45.1+ mice were transplanted with retrovirally infected hematopoietic stem cells containing human WT or R172K IDH2 mutant with an mCherry reporter and showed macrocytic anemia, thrombocytopenia, and elevated reticulocytes by 16 weeks post-transplant. In contrast to prior literature, IDH2 mutants gave rise to mature reporter positive monocytes, macrophages, and neutrophils in the bone marrow and peripheral blood. As expected, we observed myeloid skewed hematopoiesis, a trademark of MDS. When cultured ex vivo, there was loss of efferocytic capacity in IDH2 mutant bone marrow macrophages when fed apoptotic neutrophils. We then generated a genetic murine model using the Vav1 Cre IDH2 R140Q floxed mutant. The genetic model allowed us to study disease progression in the absence of radiation induced stress, and to observe mature myeloid populations while clinically defined MDS was absent. We performed ATACseq and bulk RNAseq analysis on bone marrow macrophages isolated from 3-month-old and 6-month-old mice, which demonstrated a significant decrease in global chromosomal accessibility; however, this had limited impact on the transcriptional program. Based on existing literature, the majority of macrophages in the bone marrow are in a poised state and have not yet activated genes associated with pro-efferocytic/pro-resolution or pro-inflammatory phenotypes. Since aKG is accumulated in macrophages during polarization toward reparative and pro-efferocytic phenotypes and is necessary for metabolic reprogramming for oxidative phosphorylation, we hypothesized that, in the presence of IDH2 mutations, which drain aKG from the system, pro-efferocytic polarized macrophages would be metabolically stunned. To test this theory, we isolated bone marrow macrophages from IDH2 mutants and cultured them to generate pro-efferocytic polarized macrophages. We then tested their efferocytic potential and metabolic capacity by seahorse assay. Continual efferocytosis, a hallmark of pro-efferocytic macrophages, was impaired in IDH2 mutant macrophages as early as 2-3 months of age. Consistent with this finding, mito stress testing by seahorse demonstrated that IDH2 mutant macrophages had severely impaired maximal respiratory capacity compared to WT macrophages. Treatment with global hypomethylating agent azacytidine or a cell permeable aKG analog were unable to restore respiratory capacity or efferocytic activity in the IDH2 mutant. These data strongly suggest that the efferocytic defect in IDH2 mutant macrophages is due to metabolic rather than epigenetic reprogramming. In summary, we identify defects in efferocytic function and oxidative phosphorylation in IDH2 mutant macrophages. This represents a novel mechanism that could alter the bone marrow microenvironment and impact progression of MDS and leukemic transformation. Furthermore, our data suggest that the metabolic dysregulation induced by IDH mutations may play a greater role than epigenetic impairment in IDH2 mutant mature myeloid cells, which could explain why approaches targeting hypermethylation have shown limited efficacy in clinical settings.