Neutrophil Function Research Articles

Neutrophil-Derived Proteases in Lung Inflammation: Old Players and New Prospects

Neutrophil-derived proteases are critical to the pathology of many inflammatory lung diseases, both chronic and acute. These abundant enzymes play roles in key neutrophil functions, such as neutrophil extracellular trap formation and reactive oxygen species release. They may also be released, inducing tissue damage and loss of tissue function. Historically, the neutrophil serine proteases (NSPs) have been the main subject of neutrophil protease research. Despite highly promising cell-based and animal model work, clinical trials involving the inhibition of NSPs have shown mixed results in lung disease patients. As such, the cutting edge of neutrophil-derived protease research has shifted to proteases that have had little-to-no research in neutrophils to date. These include the cysteine and serine cathepsins, the metzincins and the calpains, among others. This review aims to outline the previous work carried out on NSPs, including the shortcomings of some of the inhibitor-orientated clinical trials. Our growing understanding of other proteases involved in neutrophil function and neutrophilic lung inflammation will then be discussed. Additionally, the potential of targeting these more obscure neutrophil proteases will be highlighted, as they may represent new targets for inhibitor-based treatments of neutrophil-mediated lung inflammation.

The role of cathelicidins in neutrophil biology.

Despite their relatively short lifespan, neutrophils are tasked with counteracting pathogens through various functions, including phagocytosis, production of reactive oxygen species (ROS), neutrophil extracellular traps (NETs), and host defence peptides. Regarding the latter, small cationic cathelicidins present a conundrum in neutrophil function. Although primarily recognized as microbicides with an ability to provoke pores in microbial cell walls, the ability of cathelicidin to modulate key neutrophil functions is also of great importance, including the release of chemo-attractants, cytokines and ROS, plus prolonging neutrophil lifespan. Cumulative evidence indicates a less recognized role of cathelicidin as an "immunomodulator;" however, this term is not always explicit and its relevance in neutrophil responses during infection and inflammation is seldom discussed. This review compiles and discusses studies of how neutrophils use cathelicidin to respond to infections, while also acknowledging immunomodulatory aspects of cathelicidin through potential crosstalk between sources of the peptide.

Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells

Coronaviruses (CoVs) are enveloped single-stranded RNA viruses that predominantly attack the human respiratory system. In recent decades, several deadly human CoVs, including SARS-CoV, SARS-CoV-2, and MERS-CoV, have brought great impact on public health and economics. However, their high infectivity and the demand for high biosafety level facilities restrict the pathogenesis research of CoV infection. Exacerbated inflammatory cell infiltration is associated with poor prognosis in CoV-associated diseases. In this study, we used human CoV 229E (HCoV-229E), a CoV associated with relatively fewer biohazards, to investigate the pathogenesis of CoV infection and the regulation of neutrophil functions by CoV-infected lung cells. Induced pluripotent stem cell (iPSC)-derived alveolar epithelial type II cells (iAECIIs) exhibiting specific biomarkers and phenotypes were employed as an experimental model for CoV infection. After infection, the detection of dsRNA, S, and N proteins validated the infection of iAECIIs with HCoV-229E. The culture medium conditioned by the infected iAECIIs promoted the migration of neutrophils as well as their adhesion to the infected iAECIIs. Cytokine array revealed the elevated secretion of cytokines associated with chemotaxis and adhesion into the conditioned media from the infected iAECIIs. The importance of IL-8 secretion and ICAM-1 expression for neutrophil migration and adhesion, respectively, was demonstrated by using neutralizing antibodies. Moreover, next-generation sequencing analysis of the transcriptome revealed the upregulation of genes associated with cytokine signaling. To summarize, we established an in vitro model of CoV infection that can be applied for the study of the immune system perturbations during severe coronaviral disease.

Factors Affecting Neutrophil Functions During Sepsis: Human Microbiome and Epigenetics.

Sepsis is a severe disease that occurs when the body's immune system reacts excessively to infection. The body's response, which includes an intense anti-bacterial reaction, can damage its tissues and organs. Neutrophils are the major components of white blood cells in circulation and play a vital role in innate immunity while fighting against infections, and are considered a feature determining sepsis classification. There's a plethora of basic research detailing neutrophil functioning, among which, the study of neutrophil extracellular traps (NETs) is providing novel insights into mechanisms and treatments of sepsis. This review explores their functions, dysfunctions, and influences in the context of sepsis. The interplay between neutrophils and the human microbiome and the impact of DNA methylation on neutrophil function in sepsis are crucial areas of study. The interaction between neutrophils and the human microbiome is complex, particularly in the context of sepsis where dysbiosis may occur. We highlight the importance of deciphering neutrophil's functional alterations and their epigenetic features in sepsis because it is critical for defining sepsis endotypes and opening up the possibility for novel diagnostic methods and therapy. Specifically, epigenetic signatures are pivotal since they will provide a novel implication for sepsis diagnostic method when used in combination with the cell-free DNA (cfDNA). Research is exploring how specific patterns of DNA methylation in neutrophils, detectable in cfDNA, could serve as biomarkers for the early detection of sepsis.

Neutrophils Expressing Programmed Death-Ligand 1 Play an Indispensable Role in Effective Bacterial Elimination and Resolving Inflammation in Methicillin-Resistant Staphylococcus aureus Infection.

Programmed death ligand 1 (PD-L1) is a co-inhibitory molecule expressed on the surface of various cell types and known for its suppressive effect on T cells through its interaction with PD-1. Neutrophils also express PD-L1, and its expression is elevated in specific situations; however, the immunobiological role of PD-L1+ neutrophils has not been fully characterized. Here, we report that PD-L1-expressing neutrophils increased in methicillin-resistant Staphylococcus aureus (MRSA) infection are highly functional in bacterial elimination and supporting inflammatory resolution. The frequency of PD-L1+ neutrophils was dramatically increased in MRSA-infected mice, and this population exhibited enhanced activity in bacterial elimination compared to PD-L1- neutrophils. The administration of PD-L1 monoclonal antibody did not impair PD-L1+ neutrophil function, suggesting that PD-L1 expression itself does not influence neutrophil activity. However, PD-1/PD-L1 blockade significantly delayed liver inflammation resolution in MRSA-infected mice, as indicated by their increased plasma alanine transaminase (ALT) levels and frequencies of inflammatory leukocytes in the liver, implying that neutrophil PD-L1 suppresses the inflammatory response of these cells during the acute phase of MRSA infection. Our results reveal that elevated PD-L1 expression can be a marker for the enhanced anti-bacterial function of neutrophils. Moreover, PD-L1+ neutrophils are an indispensable population attenuating inflammatory leukocyte activities, assisting in a smooth transition into the resolution phase in MRSA infection.

Lipid mediators in neutrophil biology: inflammation, resolution and beyond.

Acute inflammation is the body's first defense in response to pathogens or injury. Failure to efficiently resolve the inflammatory insult can severely affect tissue homeostasis, leading to chronic inflammation. Neutrophils play a pivotal role in eradicating infectious pathogens, orchestrating the initiation and resolution of acute inflammation, and maintaining physiological functions. The resolution of inflammation is a highly orchestrated biochemical process, partially modulated by a novel class of endogenous lipid mediators known as specialized pro-resolving mediators (SPMs). SPMs mediate their potent bioactions via activating specific cell-surface G protein-coupled receptors (GPCR). This review focuses on recent advances in understanding the multifaceted functions of SPMs, detailing their roles in expediting neutrophil apoptosis, promoting clearance by macrophages, regulating their excessive infiltration at inflammation sites, orchestrating bone marrow deployment, also enhances neutrophil phagocytosis and tissue repair mechanisms under both physiological and pathological conditions. We also focus on the novel role of SPMs in regulating bone marrow neutrophil functions, differentiation, and highlight open questions about SPMs' functions in neutrophil heterogeneity. SPMs play a pivotal role in mitigating excessive neutrophil infiltration and hyperactivity within pathological milieus, notably in conditions such as sepsis, cardiovascular disease, ischemic events, and cancer. This significant function highlights SPMs as promising therapeutic agents in the management of both acute and chronic inflammatory disorders.

Identification of early coagulation changes associated with survival outcomes post severe burns from multiple perspectives

Coagulation alterations manifest early after severe burns and are closely linked to mortality outcomes. Nevertheless, the precise characterization of coagulation changes associated with early mortality remains elusive. We examined alterations in indicators linked to mortality outcomes at both the transcriptomic and clinical characteristic levels. At the transcriptomic level, we pinpointed 28 differentially expressed coagulation-related genes (DECRGs) following burn injuries and endeavored to validate their causal relationships through Mendelian randomization. DECRGs tied to survival exhibit a significant association with neutrophil function, wherein the expression of CYP4F2 and P2RX1 serves as robust predictors of fatal outcomes. In terms of clinical indicators, early levels of D-dimer and alterations in serum calcium show a strong correlation with mortality outcomes. Coagulation depletion and fibrinolytic activation, stemming from the hyperactivation of coagulation pathways post-severe burns, are strongly linked to patient mortality. Monitoring these early coagulation markers with predictive value can effectively identify individuals necessitating priority critical care.

Differential regulation of lung homeostasis and silicosis by the TAM receptors MerTk and Axl.

TAM receptor-mediated efferocytosis plays an important function in immune regulation and may contribute to antigen tolerance in the lungs, a site with continuous cellular turnover and generation of apoptotic cells. Some studies have identified failures in efferocytosis as a common driver of inflammation and tissue destruction in lung diseases. Our study is the first to characterize the in vivo function of the TAM receptors, Axl and MerTk, in the innate immune cell compartment, cytokine and chemokine production, as well as the alveolar macrophage (AM) phenotype in different settings in the airways and lung parenchyma. We employed MerTk and Axl defective mice to induce acute silicosis by a single exposure to crystalline silica particles (20 mg/50 μL). Although both mRNA levels of Axl and MerTk receptors were constitutively expressed by lung cells and isolated AMs, we found that MerTk was critical for maintaining lung homeostasis, whereas Axl played a role in the regulation of silica-induced inflammation. Our findings imply that MerTk and Axl differently modulated inflammatory tone via AM and neutrophil recruitment, phenotype and function by flow cytometry, and TGF-β and CXCL1 protein levels, respectively. Finally, Axl expression was upregulated in both MerTk-/- and WT AMs, confirming its importance during inflammation. This study provides strong evidence that MerTk and Axl are specialized to orchestrate apoptotic cell clearance across different circumstances and may have important implications for the understanding of pulmonary inflammatory disorders as well as for the development of new approaches to therapy.

Fracture haematoma proteomics.

The aim of this study was to determine the fracture haematoma (fxH) proteome after multiple trauma using label-free proteomics, comparing two different fracture treatment strategies. A porcine multiple trauma model was used in which two fracture treatment strategies were compared: early total care (ETC) and damage control orthopaedics (DCO). fxH was harvested and analyzed using liquid chromatography-tandem mass spectrometry. Per group, discriminating proteins were identified and protein interaction analyses were performed to further elucidate key biomolecular pathways in the early fracture healing phase. The early fxH proteome was characterized by immunomodulatory and osteogenic proteins, and proteins involved in the coagulation cascade. Treatment-specific proteome alterations were observed. The fxH proteome of the ETC group showed increased expression of pro-inflammatory proteins related to, among others, activation of the complement system, neutrophil functioning, and macrophage activation, while showing decreased expression of proteins related to osteogenesis and tissue remodelling. Conversely, the fxH proteome of the DCO group contained various upregulated or exclusively detected proteins related to tissue regeneration and remodelling, and proteins related to anti-inflammatory and osteogenic processes. The early fxH proteome of the ETC group was characterized by the expression of immunomodulatory, mainly pro-inflammatory, proteins, whereas the early fxH proteome of the DCO group was more regenerative and osteogenic in nature. These findings match clinical observations, in which enhanced surgical trauma after multiple trauma causes dysbalanced inflammation, potentially leading to reduced tissue regeneration, and gained insights into regulatory mechanisms of fracture healing after severe trauma.

Susceptibility of Melanoma Cells to Targeted Therapy Correlates with Protection by Blood Neutrophils.

Elevated levels of peripheral blood and tumor tissue neutrophils are associated with poorer clinical response and therapy resistance in melanoma. The underlying mechanism and the role of neutrophils in targeted therapy is still not fully understood. Serum samples of patients with advanced melanoma were collected and neutrophil-associated serum markers were measured and correlated with response to targeted therapy. Blood neutrophils from healthy donors and patients with advanced melanoma were isolated, and their phenotypes, as well as their in vitro functions, were compared. In vitro functional tests were conducted through nonadherent cocultures with melanoma cells. Protection of melanoma cell lines by neutrophils was assessed under MAPK inhibition. Blood neutrophils from advanced melanoma patients exhibited lower CD16 expression compared to healthy donors. In vitro, both healthy-donor- and patient-derived neutrophils prevented melanoma cell apoptosis upon dual MAPK inhibition. The effect depended on cell-cell contact and melanoma cell susceptibility to treatment. Interference with protease activity of neutrophils prevented melanoma cell protection during treatment in cocultures. The negative correlation between neutrophils and melanoma outcomes seems to be linked to a protumoral function of neutrophils. In vitro, neutrophils exert a direct protective effect on melanoma cells during dual MAPK inhibition. This study further hints at a crucial role of neutrophil-related protease activity in protection.

BTK drives neutrophil activation for sterilizing antifungal immunity.

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

Neutrophil specific glycogen metabolism supports antifungal function in the infected tissues

Abstract Neutrophils are critical for controlling systemic candidiasis (candidiasis), a bloodstream infection caused by Candida albicans. Neutrophils rely on extracellular glucose to fuel their antifungal activities at tissues. However, C. albicans consumes glucose, making it challenging for the neutrophils to survive and function at infected tissues. Hence, encountering fungal pathogens in glucose-depleted tissues exert metabolic challenges for neutrophils. It is unknown how neutrophils dynamically reprogram their metabolic pathways to function in glucose-deprived tissues in candidiasis. We show that neutrophils activate PYGL, an enzyme essential for the breakdown of intracellular glycogen storage to glucose, when glucose supply is limited. C. albicans regulate glycogenolysis in neutrophil in a dectin-1/Syk/Ca2+/PKA-axis dependent manner. Blockade of PYGL function in neutrophils inhibits fungicidal activities only when glucose availability is low and neutrophil specific PYGL ablated mice show increased susceptibility to candidiasis due to impaired ROS formation. Finally, treatment with β2-adrenergic receptor agonist, an approved drug and activator of PYGL function, improved antifungal activities of neutrophils in candidiasis. These results provide insights on how glycogenolysis supports compensatory metabolic reprograming required for antifungal function of neutrophils in the glucose-depleted infected tissues, a knowledge that can be therapeutically exploited by an approved drug.

Amyotrophic lateral sclerosis is associated with changes in peripheral neutrophil heterogeneity and function

Abstract There is overwhelming evidence that the immune system plays a significant role in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease characterized by motor neuron degeneration. Expected survival is 2-4 years from disease onset and treatment options are limited. There is significant clinical and pathogenic heterogeneity and an incomplete understanding of the underlying pathogenesis, including mechanisms of immune contribution. Neutrophils are consistently implicated in ALS, with increased count in the blood and infiltration into the spinal cord. Increased neutrophil count and activation are associated with shortened survival and neutrophil levels are the single best predictor of future ALS progression. Despite this, little is known about which neutrophil subpopulations are dysregulated or the underlying mechanisms contributing to disease progression. Primary neutrophils were assessed via flow and ex vivo stimulation. NET remnants (MPO:DNA, elastase, extracellular DNA) were quantitated in plasma. Distinct populations were observed in controls and ALS subjects, and ex vivo analysis of primary neutrophils and analysis of NET remnants demonstrates changes in effector functions that may contribute to BBB disruption and neuronal damage in ALS. These differences in neutrophil populations and function provide compelling evidence that neutrophils play a role in ALS progression, as well as provide novel targets for therapeutics.

Sphingosine kinase 2 suppresses neutrophil responses while attenuating immune pathology during a persistent viral infection

Abstract During chronic virus infections, the functions of immune cells are often suppressed which may restrict immune pathology but lead to viral persistence. However, the underlying mechanisms are incompletely understood. Sphingosine kinase 2 (SphK2) generates sphingosine 1-phosphate, a bioactive lipid mediator, and mediates diverse cellular processes. We have recently found that SphK2-deficient mice succumb to infection by lymphocytic choriomeningitis virus (LCMV) clone 13 (Cl 13) due to immune pathology associated with nephropathy. Heightened T cell immunity was crucial for infection-induced mortality of SphK2 KO mice. However, in addition to T cell immunity, a notable increase of neutrophils was observed in multiple tissues as well as blood of LCMV-infected SphK2 KO mice. Importantly, SphK2-deficient neutrophils expressed substantially lower levels of the immune suppressive marker CD244 and produced higher amounts of ROS compared to SphK2-sufficient neutrophils upon infection. Depletion of neutrophils from SphK2 KO mice increased the viability of infected mice. Further, adoptively transferred SphK2-deficient neutrophils improved virus-specific T cell responses in wild type mice when compared to the transferred SphK2-sufficient neutrophils. The results indicate that SphK2 inhibits neutrophil expansion and function, contributing to the immune regulation during a persistent virus infection. The study may help to design new immune therapeutics to control viral diseases.

Effects of Exercise Therapy on Circadian Rhythm Regulation of Neutrophils

Abstract Background: Physical inactivity is recognized as a risk factor for numerous common illnesses, while exercise has been shown to be beneficial. Neutrophils are the first line of cell-mediated defense, with characterized by circadian rhythms. The liver is metabolically active organ that is strongly influenced by circadian regulation. The effects of exercise-induced circadian changes may modify the neutrophils phenotype and function. Methods: Male mice undergoing a four-week treadmill regimen for 1h at ZT3(3h after lights on). Bone marrow, blood and liver tissue were analyzed at different time points. Circadian genes and neutrophil subpopulations were assessed. Results: The circulating neutrophil numbers were fluctuated according to a circadian pattern with a peak at ZT3 and a nadir at ZT15. Rhythmicity analysis revealed that the circadian amplitude was increased, but the period and phase were unaltered. Mice subjected to exercise had fewer immature and higher mature neutrophils in bone marrow at ZT3. The expression of clock-related genes in liver, including Arntl and Clock, increased at ZT3, while others, like Per1 were decreased. The number of aged neutrophil follows diurnal patterns with a peak at ZT3, and exhibited a greater percentage and number of neutrophils with an aging phenotype in blood at Exercise group. Summary: Exercise modulates circadian-dependent neutrophils quiescence, promotes maturation of myeloid neutrophils and driven circulating neutrophil aging phenotype.

Highly Motile Neutrophil Cytoplast ameliorate Bacterial Pneumonia

Abstract Polymorphonuclear neutrophils (PMN) function as innate immune cell through rapid mobilization at site of infection and prompt phagocytosis and bactericidal activity. We addressed in vivo in mouse lungs the function of cytoplasts (PMNcyt), the PMN generated following release of extracellular traps (NETs). We found ~50% of PMN lost their nucleus post-NETosis following pneumonia induced by Pseudomonas aeruginosa (PA), and become PMNcyt. Interestingly, , PMNcyt in lung microvessels showed >2 greater velocity and > 3 greater deformability as compared to PMN. PMNcyt were also 9x more efficient in killing airway PA as compared to PMN. Using the protein arginine deiminase 4 (PAD4) inhibitor (Cl-amidine) to prevent NET formation in mice, we prevented the generation of PMNcyt demonstrating that PMNcyt were derived post-vital NETosis. We also showed PMNcyt retained the ability to phagocytose PA. Moreover, the inhibition of PMNcyt generation by PAD4 blocker induced severe lung injury in the pneumonia model, indicating an important anti-bacterial protective role of PMNcyt. Adoptive transfer of PMNcyt in PA infected mice also significantly reduced bacterial load and prevented lung tissue injury in mice. The results suggest a crucial host-defense function of PMNcyt generated post vital NETosis and their usefulness as cell-based therapy in inflammatory diseases such as pneumonia.

N-glycan signature of neutrophil function in a Staphylococcus aureus skin infection model by mass spectrometry imaging

Abstract Staphylococcus aureus (Sa) is a public health concern due to the rise of antibiotic-resistant strains. In humans, elevated risk is associated with Tumor Necrosis Factor (TNF) inhibition. Neutrophils expressing TNF receptors (TNFR1 and TNFR2) respond to Sa skin infection in a mouse model. TNFR2 activation induces PAD4 (peptidylarginine-deiminase-4) which leads to neutrophil extracellular traps (NET) synthesis, increasing host defenses. Alterations in PAD4 and N-glycans are related to increased skin infections and the spatial glycosylation signature during the skin response to Sa is unknown. We compared N-glycan signatures in neutrophil region between wildtype (WT), TNFR1 KO, TNFR2 KO and PAD4-/- groups. Mice were intradermally injected with Sa and skin samples collected for analysis by mass spectrometry imaging. We sprayed PNGaseF on prepared FFPE sections to cleave the N-glycans, sprayed with CHCA matrix, and evaluated in positive ion mode at 50mm spatial resolution on a timsTOF Flex (Bruker Daltonics). Differential N-glycan signatures were identified using a histology-guided approach limiting the comparisons to the PMN region. PAD4-/- showed higher intensity of N-glycans than all groups. However, specific N-glycans were higher in TNFR1 KO than PAD4-/- and TNFR2. A lower profile was found in TNFR2 KO vs. WT. We concluded that each group has a specific N-glycan signature which can be attributed to unique immune responses to Sa infection and NET formation.

Abstract 2136: The Effects Of Dual Antiplatelet Therapy On Neutrophil Function

Introduction: Dual anti-platelet therapy (DAPT) is routinely administered to inhibit platelet aggregation in acute myocardial infarction (AMI) and, in doing so, reduces the risk of recurrent adverse cardiovascular events. Alongside platelets, neutrophils are also key mediators of inflammation during AMI, both secreting cytokines and chemokines and producing Neutrophil Extracellular Traps (NETs). While DAPT is known to mediate off-target anti-inflammatory effects, the extent to which DAPT affects neutrophil function is not well understood. Objective: To investigate the effects of DAPT on neutrophil-mediated inflammation. Methods: Neutrophils were isolated from whole blood of ten healthy subjects pre- and post-DAPT (ticagrelor and aspirin) treatment and NET formation in response to classical NET-inducing agonists (PMA and ionomycin) was measured by SYTOX Green fluorescence and MPO activity. Cytokine and chemokine production following stimulation with an activating agonist (FSL-1) was measured by cytometric bead array and reported as median fluorescence intensity (MFI). DAPT was effective in inhibiting platelet aggregation in response to AA and ADP. Results: NET formation in response to ionomycin, but not PMA, was greater in DAPT-treated neutrophils compared to treatment-naïve neutrophils (69.9 % ± 30.66 SEM). Levels of inflammatory mediators were also markedly different pre- and post- DAPT treatment. For example, MFI of IL-8 was decreased by 18% ± 9.97 SEM post-DAPT compared with pre-DAPT MFI, while MFI of IL-12p and IFN-α were significantly increased (32.54% ± 12.12 SEM, 58.68% ± 18.72 SEM respectively) compared to pre-DAPT. Conclusion: DAPT primes neutrophils for inducing some, but not all, NETotic pathways and releasing some, but not all, inflammatory mediators. This suggests that our primary strategy for inhibiting platelet aggregation in AMI does not provide broad off-target anti-inflammatory effects on neutrophils. In fact, DAPT may encourage a pro-inflammatory neutrophil phenotype.

Defining healthy and breast tumor-associated neutrophils at a whole-organism, single-cell resolution

Abstract Neutrophils accumulate systemically in tumor-bearing organisms, and have been shown to promote primary tumor progression and metastasis through suppression of the anti-tumor immune response. However, neutrophils have specialized phenotypes in different organs, and their full transcriptional heterogeneity and maturation trajectory has yet to be defined across healthy and cancer states. Here, we performed single-cell RNA sequencing on isolated neutrophils from bone marrow, spleen, lymph nodes, blood, mammary fat pad/tumor, and lungs from wild-type and breast tumor-bearing MMTV-PyMT mice. Our analysis reveals distinct transcriptional programs which distinguish systemic neutrophil function in normal homeostasis and cancer. In wild-type mice, we describe a neutrophil maturation trajectory that begins with granulopoiesis in the bone marrow, continues development in the spleen and blood, and reaches a fully mature state in the lymph nodes, mammary fat pad, and lungs. This is captured by five maturation stages, N0-N4, each with its own characteristic gene signature. In MMTV-PyMT mice, we observe a systemic upregulation of genes including Ifitm1, Wfdc17, Clec4e, Igfbp6, and Saac, which have been implicated in cancer-induced immunosuppression. Taken altogether, this work provides a multi-organ perspective into neutrophil development across healthy and cancer states and identifies markers specific to breast tumor-associated neutrophils which may be potential immunotherapeutic targets.

Abstract 105: Neutrophil Cracr2a Is Crucial For Calcium Signaling And Enhances Neutrophil Adhesive Function Under Thromboinflammatory Conditions

Ca 2+ release-activated channel regulator 2A (CRACR2A) is a Ca 2+ -binding protein that facilitates store-operated Ca 2+ entry during T cell activation. However, it is unknown whether neutrophil CRACR2A regulates neutrophil functions in inflammation. Our confocal intravital microscopy (IVM) revealed that compared to wild-type (WT) mice, myeloid-specific cracr2a conditional knockout (CKO, cracr2a fl/fl;Lyz-cre ) mice exhibited a significant increase in neutrophil rolling and a decrease in neutrophil adhesion, crawling, and transmigration on ear and cremaster venules in TNF-α-induced inflammation. Deletion of neutrophil cracr2a impaired β2 integrin function without affecting the expression of L-selectin, PECAM-1, and JAM-A. Neutrophil cracr2a increased Ca 2+ mobilization after stimulation with fMLP, A23187, and thapsigargin by its rapid interaction with STIM1. Due to the lack of CRACR2A inhibitors, we generated CRACR2A-deleted HL60 cells using CRISPR/Cas9. CRACR2A deletion in differentiated HL60 cells recapitulated the defects seen in cracr2a-null neutrophils, including reduced β2 integrin function and Ca 2+ mobilization after agonist stimulation. Since excessively recruited neutrophils cause brain damage in ischemic stroke, we examined whether neutrophil cracr2a contributes to stroke pathology. Using a transient middle cerebral artery occlusion (tMCAO)-induced ischemic stroke model, we found that compared to WT mice, cracr2a CKO mice exhibited improved neurological deficits and reduced infarct volume. Longitudinal 4D IVM of mouse brain revealed that neutrophil cracr2a is crucial for neutrophil adhesion and transmigration on cerebral vessels 16-24 hours after tMCAO and upregulated αMβ2 integrin on adherent neutrophils. Further, single-cell behavior analysis of 4D IVM showed that a proinflammatory subset of neutrophils arise in WT mice, but not cracr2a CKO mice, after tMCAO. We observed that the number of circulating pro-inflammatory neutrophils was markedly decreased 24 hours after tMCAO in cracr2a CKO mice compared with WT mice. Our findings indicate that neutrophil CRACR2A is a key regulator for Ca 2+ signaling and enhances neutrophil pro-inflammatory and adhesive activities in vascular inflammation and ischemic stroke.