The local microenvironment drives activation of neutrophils in human brain tumors.

Neutrophils are abundant immune cells in the circulation and frequently infiltrate tumors in substantial numbers. However, their precise functions in different cancer types remain incompletely understood, including in the brain microenvironment. We therefore investigated neutrophils in tumor tissue of glioma and brain metastasis patients, with matched peripheral blood, and herein describe the first in-depth analysis of neutrophil phenotypes and functions in these tissues. Orthogonal profiling strategies in humans and mice revealed that brain tumor-associated neutrophils (TANs) differ significantly from blood neutrophils and have a prolonged lifespan and immune-suppressive and pro-angiogenic capacity. TANs exhibit a distinct inflammatory signature, driven by a combination of soluble inflammatory mediators including tumor necrosis factor alpha (TNF-ɑ) and Ceruloplasmin, which is more pronounced in TANs from brain metastasis versus glioma. Myeloid cells, including tumor-associated macrophages, emerge at the core of this network of pro-inflammatory mediators, supporting the concept of a critical myeloid niche regulating overall immune suppression in human brain tumors.

Strategies of neutrophil diversification.

Neutrophils are formidable defenders. Their vast numbers, constant production, high cytotoxicity and capacity to produce extracellular traps, underlie their ability to efficiently protect in a microorganism-rich world. However, neutrophils are much more than immune sentinels, as evidenced by the expanding repertoire of functions discovered in the context of tissue homeostasis, regeneration or chronic pathologies. In this Perspective, we discuss general functional features of the neutrophil compartment that may be relevant in most, if not all, physiological scenarios in which they participate, including specialization in naïve tissues, transcriptional noise in the bloodstream as a potential strategy for diversification and functional bias in inflammatory sites. We intentionally present the reader with more questions than answers and propose models and approaches that we hope will shed new light onto the biology of these fascinating cells and spark new directions of research.

Co-option of Neutrophil Fates by Tissue Environments.

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands.

Distinct transcription factor networks control neutrophil-driven inflammation.

Neutrophils display distinct gene expression patters depending on their developmental stage, activation state and tissue microenvironment. To determine the transcription factor networks that shape these responses in a mouse model, we integrated transcriptional and chromatin analyses of neutrophils during acute inflammation. We showed active chromatin remodeling at two transition stages: bone marrow-to-blood and blood-to-tissue. Analysis of differentially accessible regions revealed distinct sets of putative transcription factors associated with control of neutrophil inflammatory responses. Using ex vivo and in vivo approaches, we confirmed that RUNX1 and KLF6 modulate neutrophil maturation, whereas RELB, IRF5 and JUNB drive neutrophil effector responses and RFX2 and RELB promote survival. Interfering with neutrophil activation by targeting one of these factors, JUNB, reduced pathological inflammation in a mouse model of myocardial infarction. Therefore, our study represents a blueprint for transcriptional control of neutrophil responses in acute inflammation and opens possibilities for stage-specific therapeutic modulation of neutrophil function in disease.

Neutrophil "plucking" on megakaryocytes drives platelet production and boosts cardiovascular disease.

Intravascular neutrophils and platelets collaborate in maintaining host integrity, but their interaction can also trigger thrombotic complications. We report here that cooperation between neutrophil and platelet lineages extends to the earliest stages of platelet formation by megakaryocytes in the bone marrow. Using intravital microscopy, we show that neutrophils "plucked" intravascular megakaryocyte extensions, termed proplatelets, to control platelet production. Following CXCR4-CXCL12-dependent migration towards perisinusoidal megakaryocytes, plucking neutrophils actively pulled on proplatelets and triggered myosin light chain and extracellular-signal-regulated kinase activation through reactive oxygen species. By these mechanisms, neutrophils accelerate proplatelet growth and facilitate continuous release of platelets in steady state. Following myocardial infarction, plucking neutrophils drove excessive release of young, reticulated platelets and boosted the risk of recurrent ischemia. Ablation of neutrophil plucking normalized thrombopoiesis and reduced recurrent thrombosis after myocardial infarction and thrombus burden in venous thrombosis. We establish neutrophil plucking as a target to reduce thromboischemic events.

A Neutrophil Timer Coordinates Immune Defense and Vascular Protection.

Neutrophils eliminate pathogens efficiently but can inflict severe damage to the host if they over-activate within blood vessels. It is unclear how immunity solves the dilemma of mounting an efficient anti-microbial defense while preserving vascular health. Here, we identify a neutrophil-intrinsic program that enabled both. The gene Bmal1 regulated expression of the chemokine CXCL2 to induce chemokine receptor CXCR2-dependent diurnal changes in the transcriptional and migratory properties of circulating neutrophils. These diurnal alterations, referred to as neutrophil aging, were antagonized by CXCR4 (C-X-C chemokine receptor type 4) and regulated the outer topology of neutrophils to favor homeostatic egress from blood vessels at night, resulting in boosted anti-microbial activity in tissues. Mice engineered for constitutive neutrophil aging became resistant to infection, but the persistence of intravascular aged neutrophils predisposed them to thrombo-inflammation and death. Thus, diurnal compartmentalization of neutrophils, driven by an internal timer, coordinates immune defense and vascular protection.

Role of TLR4 (Toll-Like Receptor 4) in N1/N2 Neutrophil Programming After Stroke.

Background and Purpose- After stroke, the population of infiltrated neutrophils in the brain is heterogeneous, including a population of alternative neutrophils (N2) that express M2 phenotype markers. We explored the role of TLR4 (toll-like receptor 4) on neutrophil infiltration and polarization in this setting. Methods- Focal cerebral ischemia was induced by occlusion of the middle cerebral artery occlusion in TLR4-KO and WT (wild type) mice. Infarct size was measured by Nissl staining and magnetic resonance imaging. Leukocyte infiltration was quantified 48 hours after middle cerebral artery occlusion by immunofluorescence and flow cytometry. To elucidate mechanisms underlying TLR4-mediated N2 phenotype, a cDNA microarray analysis was performed in neutrophils isolated from blood 48 hours after stroke in WT and TLR4-KO mice. Results- As demonstrated previously, TLR4-deficient mice presented lesser infarct volumes than WT mice. TLR4-deficient mice showed higher density of infiltrated neutrophils 48 hours after stroke compared with WT mice, concomitantly to neuroprotection. Furthermore, cytometric and stereological analyses revealed an increased number of N2 neutrophils (YM1+ cells) into the ischemic core in TLR4-deficient mice, suggesting a protective effect of this neutrophil subset that was corroborated by depleting peripheral neutrophils or using mice with TLR4 genetically ablated in the myeloid lineage. Finally, cDNA microarray analysis in neutrophils, confirmed by quantitative polymerase chain reaction, showed that TLR4 modulates several pathways associated with ischemia-induced inflammation, migration of neutrophils into the parenchyma, and their functional priming, which might explain the opposite effect on outcome of the different neutrophil subsets. Conclusions- TLR4 deficiency increased the levels of alternative neutrophils (N2)-an effect associated with neuroprotection after stroke-supporting that modulation of neutrophil polarization is a major target of TLR4 and highlighting the crucial role of TLR4 at the peripheral level after stroke. Visual Overview- An online visual overview is available for this article.

L-kynurenine/aryl hydrocarbon receptor pathway mediates brain damage after experimental stroke.

BACKGROUND: Aryl hydrocarbon receptor (AhR) is a transcription factor that belongs to the basic helix-loop-helix PAS (Per-Arnt-Sim homology domain) family known to mediate the toxic and carcinogenic effects of xenobiotics. Interestingly, AhR is widely expressed in the central nervous system, but its physiological and pathological roles are still unclear. METHODS AND RESULTS: To define the role of AhR in stroke, we used middle cerebral artery occlusion in mice and oxygen-glucose deprivation in rat cortical neurons. The results presented here show that the ischemic insult increases total and nuclear AhR levels and AhR transcriptional activity in neurons in vivo and in vitro. We also show that AhR has a causal role in acute ischemic damage because pharmacological or genetic loss-of-function approaches result in neuroprotection. Inhibition of cAMP response element-binding protein-dependent signaling may participate in the deleterious actions of AhR. Finally, we have also found that L-kynurenine, a tryptophan metabolite with AhR agonistic properties, is an endogenous ligand that mediates AhR activation in the brain after middle cerebral artery occlusion. CONCLUSIONS: Our data demonstrate that an L-kynurenine/AhR pathway mediates acute brain damage after stroke and open new possibilities for the diagnosis and treatment of this pathology.

Aging increases microglial proliferation, delays cell migration, and decreases cortical neurogenesis after focal cerebral ischemia.

BACKGROUND: Aging is not just a risk factor of stroke, but it has also been associated with poor recovery. It is known that stroke-induced neurogenesis is reduced but maintained in the aged brain. However, there is no consensus on how neurogenesis is affected after stroke in aged animals. Our objective is to determine the role of aging on the process of neurogenesis after stroke. METHODS: We have studied neurogenesis by analyzing proliferation, migration, and formation of new neurons, as well as inflammatory parameters, in a model of cerebral ischemia induced by permanent occlusion of the middle cerebral artery in young- (2 to 3 months) and middle-aged mice (13 to 14 months). RESULTS: Aging increased both microglial proliferation, as shown by a higher number of BrdU(+) cells and BrdU/Iba1(+) cells in the ischemic boundary and neutrophil infiltration. Interestingly, aging increased the number of M1 monocytes and N1 neutrophils, consistent with pro-inflammatory phenotypes when compared with the alternative M2 and N2 phenotypes. Aging also inhibited (subventricular zone) SVZ cell proliferation by decreasing both the number of astrocyte-like type-B (prominin-1(+)/epidermal growth factor receptor (EGFR)(+)/nestin(+)/glial fibrillary acidic protein (GFAP)(+) cells) and type-C cells (prominin-1(+)/EGFR(+)/nestin(-)/Mash1(+) cells), and not affecting apoptosis, 1 day after stroke. Aging also inhibited migration of neuroblasts (DCX(+) cells), as indicated by an accumulation of neuroblasts at migratory zones 14 days after injury; consistently, aged mice presented a smaller number of differentiated interneurons (NeuN(+)/BrdU(+) and GAD67(+) cells) in the peri-infarct cortical area 14 days after stroke. CONCLUSIONS: Our data confirm that stroke-induced neurogenesis is maintained but reduced in aged animals. Importantly, we now demonstrate that aging not only inhibits proliferation of specific SVZ cell subtypes but also blocks migration of neuroblasts to the damaged area and decreases the number of new interneurons in the cortical peri-infarct area. Thus, our results highlight the importance of using aged animals for translation to clinical studies.

Variable selection for nonlinear dimensionality reduction of biological datasets through bootstrapping of correlation networks.

Identifying the most relevant variables or features in massive datasets for dimensionality reduction can lead to improved and more informative display, faster computation times, and more explainable models of complex systems. Despite significant advances and available algorithms, this task generally remains challenging, especially in unsupervised settings. In this work, we propose a method that constructs correlation networks using all intervening variables and then selects the most informative ones based on network bootstrapping. The method can be applied in both supervised and unsupervised scenarios. We demonstrate its functionality by applying Uniform Manifold Approximation and Projection for dimensionality reduction to several high-dimensional biological datasets, derived from 4D live imaging recordings of hundreds of morpho-kinetic variables, describing the dynamics of thousands of individual leukocytes at sites of prominent inflammation. We compare our method with other standard ones in the field, such as Principal Component Analysis and Elastic Net, showing that it outperforms them. The proposed method can be employed in a wide range of applications, encompassing data analysis and machine learning.

Deterministic reprogramming of neutrophils within tumors.

Neutrophils are increasingly recognized as key players in the tumor immune response and are associated with poor clinical outcomes. Despite recent advances characterizing the diversity of neutrophil states in cancer, common trajectories and mechanisms governing the ontogeny and relationship between these neutrophil states remain undefined. Here, we demonstrate that immature and mature neutrophils that enter tumors undergo irreversible epigenetic, transcriptional, and proteomic modifications to converge into a distinct, terminally differentiated dcTRAIL-R1+ state. Reprogrammed dcTRAIL-R1+ neutrophils predominantly localize to a glycolytic and hypoxic niche at the tumor core and exert pro-angiogenic function that favors tumor growth. We found similar trajectories in neutrophils across multiple tumor types and in humans, suggesting that targeting this program may provide a means of enhancing certain cancer immunotherapies.

N2 neutrophils, novel players in brain inflammation after stroke: modulation by the PPARγ agonist rosiglitazone.

BACKGROUND AND PURPOSE: Neutrophils have been traditionally recognized as major mediators of a deleterious inflammatory response in acute ischemic stroke, but their potential as a therapeutic target remains unexplored. Recent evidence indicates that neutrophils may acquire different phenotypes and contribute to resolution of inflammation through the release of anti-inflammatory mediators. Thus, similar to M2 macrophages, neutrophils have been proposed to shift toward an N2 phenotype, a polarization that is peroxisome proliferator-activated receptor-γ dependent in macrophages. We hypothesize that peroxisome proliferator-activated receptor-γ activation with rosiglitazone induces changes in neutrophilic mobilization and phenotype that might influence stroke outcome. METHODS: Brain sections and cell suspensions were prepared from mice exposed to permanent distal middle cerebral artery occlusion. Double immunostaining with stereological counting of brain sections and flow-cytometry analysis of brain cell suspensions were performed. RESULTS: Rosiglitazone accelerated neutrophil infiltration to the ischemic core, concomitantly to neuroprotection. Some neutrophils (≈31%) expressed M2 markers, namely Ym1 and CD206 (mannose receptor). After treatment with the peroxisome proliferator-activated receptor-γ agonist rosiglitazone, most neutrophils (≈77%) acquired an N2 phenotype. Interestingly, rosiglitazone increased neutrophil engulfment by microglia/macrophages, a clearance that preferentially affected the N2 subset. CONCLUSIONS: We present the first evidence of neutrophil reprogramming toward an N2 phenotype in brain inflammation, which can be modulated by activation of the peroxisome proliferator-activated receptor-γ nuclear receptor. We also show that N2 polarization is associated with an increased neutrophil clearance, thus suggesting that this switch is a crucial event for resolution of inflammation that may participate in neuroprotection.

Silent information regulator 1 protects the brain against cerebral ischemic damage.

BACKGROUND AND PURPOSE: Sirtuin 1 (SIRT1) is a member of NAD+-dependent protein deacetylases implicated in a wide range of cellular functions and has beneficial properties in pathologies including ischemia/reperfusion processes and neurodegeneration. However, no direct evidence has been reported on the direct implication of SIRT1 in ischemic stroke. The aim of this study was to establish the role of SIRT1 in stroke using an experimental model in mice. METHODS: Wild-type and Sirt1-/- mice were subjected to permanent focal ischemia by permanent ligature. In another set of experiments, wild-type mice were treated intraperitoneally with vehicle, activator 3 (SIRT1 activator, 10 mg/kg), or sirtinol (SIRT1 inhibitor, 10 mg/kg) for 10 minutes, 24 hours, and 40 hours after ischemia. Brains were removed 48 hours after ischemia for determining the infarct volume. Neurological outcome was evaluated using the modified neurological severity score. RESULTS: Exposure to middle cerebral artery occlusion increased SIRT1 expression in neurons of the ipsilesional mouse brain cortex. Treatment of mice with activator 3 reduced infarct volume, whereas sirtinol increased ischemic injury. Sirt1-/- mice displayed larger infarct volumes after ischemia than their wild-type counterparts. In addition, SIRT1 inhibition/deletion was concomitant with increased acetylation of p53 and nuclear factor κB (p65). CONCLUSIONS: These results support the idea that SIRT1 plays an important role in neuroprotection against brain ischemia by deacetylation and subsequent inhibition of p53-induced and nuclear factor κB-induced inflammatory and apoptotic pathways.

Citicoline (CDP-choline) increases Sirtuin1 expression concomitant to neuroprotection in experimental stroke.

CDP-choline has shown neuroprotective effects in cerebral ischemia. In humans, although a recent trial International Citicoline Trial on Acute Stroke (ICTUS) has shown that global recovery is similar in CDP-choline and placebo groups, CDP-choline was shown to be more beneficial in some patients, such as those with moderate stroke severity and not treated with t-PA. Several mechanisms have been proposed to explain the beneficial actions of CDP-choline. We have now studied the participation of Sirtuin1 (SIRT1) in the neuroprotective actions of CDP-choline. Fischer rats and Sirt1⁻/⁻ mice were subjected to permanent focal ischemia. CDP-choline (0.2 or 2 g/kg), sirtinol (a SIRT1 inhibitor; 10 mg/kg), and resveratrol (a SIRT1 activator; 2.5 mg/kg) were administered intraperitoneally. Brains were removed 24 and 48 h after ischemia for western blot analysis and infarct volume determination. Treatment with CDP-choline increased SIRT1 protein levels in brain concomitantly to neuroprotection. Treatment with sirtinol blocked the reduction in infarct volume caused by CDP-choline, whereas resveratrol elicited a strong synergistic neuroprotective effect with CDP-choline. CDP-choline failed to reduce infarct volume in Sirt1⁻/⁻ mice. Our present results demonstrate a robust effect of CDP-choline like SIRT1 activator by up-regulating its expression. Our findings suggest that therapeutic strategies to activate SIRT1 may be useful in the treatment of stroke. Sirtuin 1 (SIRT1) is implicated in a wide range of cellular functions. Regarding stroke, there is no direct evidence. We have demonstrated that citicoline increases SIRT1 protein levels in brain concomitantly to neuroprotection. Citicoline fails to reduce infarct volume in Sirt1⁻/⁻ mice. Our findings suggest that therapeutic strategies acting on SIRT1 may be useful in the treatment of stroke.

Neutrophils in cancer, a love-hate affair.

Neutrophils dominate the immunological landscape of multiple types of solid tumours in mice and humans and exert different pro- or antitumoral activity. This functional heterogeneity has prompted a search for different subsets and classifications of tumour-infiltrating neutrophils with the idea of better delineating their specific roles in cancer. In this review, we describe current studies that highlight specific mechanisms by which neutrophils exert pro- or antitumoral function and focus on how distinct tumour types induce unique functional states in neutrophils, co-opt granulopoiesis, modulate neutrophil ageing and prolong the neutrophil life span. In addition, we discuss how the tissue-specific tumour stroma and the stage of the cancer influence the function and number of tumour-infiltrating neutrophils. Finally, we explore different approaches to enhance the therapeutic efficacy in cancer types dominated by neutrophils.

Understanding NSCLC, one cell at a time.

Lung cancers are very heterogeneous, a feature that in part emanates from the tumor microenvironment. In this issue, Salcher et al. provide a comprehensive analysis of hundreds of patients with non-small cell lung cancer (NSCLC) at the single-cell level to discover extreme immune diversity and define neutrophil populations associated with treatment outcome.

How to bridle a neutrophil.

Recent high-dimensional technologies have enabled the characterization of heterogeneity in the neutrophil compartment at an unprecedented resolution. In this review, we discuss the emerging notion of heterogeneity within the neutrophil pool, and provide a detailed account of evolving concepts in the field. We place special focus on neutrophil differentiation in the bone marrow and plasticity in tissues, describe the limitations that arise when exploring neutrophil heterogeneity using single-cell analyses, and suggest state-of-the-art alternatives to improve their characterization. Finally, we propose strategies arising from these new concepts that may allow us to bridle neutrophil plasticity towards therapeutic benefit.

Combined statistical modeling enables accurate mining of circadian transcription.

Circadian-regulated genes are essential for tissue homeostasis and organismal function, and are therefore common targets of scrutiny. Detection of rhythmic genes using current analytical tools requires exhaustive sampling, a demand that is costly and raises ethical concerns, making it unfeasible in certain mammalian systems. Several non-parametric methods have been commonly used to analyze short-term (24 h) circadian data, such as JTK_cycle and MetaCycle. However, algorithm performance varies greatly depending on various biological and technical factors. Here, we present CircaN, an ad-hoc implementation of a non-linear mixed model for the identification of circadian genes in all types of omics data. Based on the variable but complementary results obtained through several biological and in silico datasets, we propose a combined approach of CircaN and non-parametric models to dramatically improve the number of circadian genes detected, without affecting accuracy. We also introduce an R package to make this approach available to the community.