Hypoxia-inducible factor orchestrates adenosine metabolism to promote liver cancer development.

Hypoxia-induced adenosine creates an immunosuppressive tumor microenvironment (TME) and dampens the efficacy of immune checkpoint inhibitors (ICIs). We found that hypoxia-inducible factor 1 (HIF-1) orchestrates adenosine efflux through two steps in hepatocellular carcinoma (HCC). First, HIF-1 activates transcriptional repressor MXI1, which inhibits adenosine kinase (ADK), resulting in the failure of adenosine phosphorylation to adenosine monophosphate. This leads to adenosine accumulation in hypoxic cancer cells. Second, HIF-1 transcriptionally activates equilibrative nucleoside transporter 4, pumping adenosine into the interstitial space of HCC, elevating extracellular adenosine levels. Multiple in vitro assays demonstrated the immunosuppressive role of adenosine on T cells and myeloid cells. Knockout of ADK in vivo skewed intratumoral immune cells to protumorigenic and promoted tumor progression. Therapeutically, combination treatment of adenosine receptor antagonists and anti-PD-1 prolonged survival of HCC-bearing mice. We illustrated the dual role of hypoxia in establishing an adenosine-mediated immunosuppressive TME and offered a potential therapeutic approach that synergizes with ICIs in HCC.

Ferroptosis Suppressor Protein 1 Inhibition Promotes Tumor Ferroptosis and Anti-tumor Immune Responses in Liver Cancer.

BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with dreadful clinical outcome. Tyrosine kinase inhibitors and immune checkpoint inhibitors are the only United States Food and Drug Administration-approved therapeutic options for patients with advanced HCC with limited therapeutic success. Ferroptosis is a form of immunogenic and regulated cell death caused by chain reaction of iron-dependent lipid peroxidation. Coenzyme Q10 (CoQ10)/ferroptosis suppressor protein 1 (FSP1) axis was recently identified as a novel protective mechanism against ferroptosis. We would like to explore whether FSP1 could be a potential therapeutic target for HCC. METHODS: FSP1 expression in human HCC and paired non-tumorous tissue samples were determined by reverse transcription-quantitative polymerase chain reaction, followed by clinicopathologic correlation and survival studies. Regulatory mechanism for FSP1 was determined using chromatin immunoprecipitation. The hydrodynamic tail vein injection model was used for HCC induction to evaluate the efficacy of FSP1 inhibitor (iFSP1) in vivo. Single-cell RNA sequencing revealed the immunomodulatory effects of iFSP1 treatment. RESULTS: We showed that HCC cells greatly rely on the CoQ10/FSP1 system to overcome ferroptosis. We found that FSP1 was significantly overexpressed in human HCC and is regulated by kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 pathway. FSP1 inhibitor iFSP1 effectively reduced HCC burden and profoundly increased immune infiltrates including dendritic cells, macrophages, and T cells. We also demonstrated that iFSP1 worked synergistically with immunotherapies to suppress HCC progression. CONCLUSIONS: We identified FSP1 as a novel, vulnerable therapeutic target in HCC. The inhibition of FSP1 potently induced ferroptosis, which promoted innate and adaptive anti-tumor immune responses and effectively suppressed HCC tumor growth. FSP1 inhibition therefore represents a new therapeutic strategy for HCC.

Polo-like kinase 4 inhibitor CFI-400945 suppresses liver cancer through cell cycle perturbation and eliciting antitumor immunity.

BACKGROUND AND AIMS: Prognosis of HCC remains poor due to lack of effective therapies. Immune checkpoint inhibitors (ICIs) have delayed response and are only effective in a subset of patients. Treatments that could effectively shrink the tumors within a short period of time are idealistic to be employed together with ICIs for durable tumor suppressive effects. HCC acquires increased tolerance to aneuploidy. The rapid division of HCC cells relies on centrosome duplication. In this study, we found that polo-like kinase 4 (PLK4), a centrosome duplication regulator, represents a therapeutic vulnerability in HCC. APPROACH AND RESULTS: An orally available PLK4 inhibitor, CFI-400945, potently suppressed proliferating HCC cells by perturbing centrosome duplication. CFI-400945 induced endoreplication without stopping DNA replication, causing severe aneuploidy, DNA damage, micronuclei formation, cytosolic DNA accumulation, and senescence. The cytosolic DNA accumulation elicited the DEAD box helicase 41-stimulator of interferon genes-interferon regulatory factor 3/7-NF-κß cytosolic DNA sensing pathway, thereby driving the transcription of senescence-associated secretory phenotypes, which recruit immune cells. CFI-400945 was evaluated in liver-specific p53/phosphatase and tensin homolog knockout mouse HCC models established by hydrodynamic tail vein injection. Tumor-infiltrated immune cells were analyzed. CFI-400945 significantly impeded HCC growth and increased infiltration of cluster of differentiation 4-positive (CD4 + ), CD8 + T cells, macrophages, and natural killer cells. Combination therapy of CFI-400945 with anti-programmed death-1 showed a tendency to improve HCC survival. CONCLUSIONS: We show that by targeting a centrosome regulator, PLK4, to activate the cytosolic DNA sensing-mediated immune response, CFI-400945 effectively restrained tumor progression through cell cycle inhibition and inducing antitumor immunity to achieve a durable suppressive effect even in late-stage mouse HCC.

Using mouse liver cancer models based on somatic genome editing to predict immune checkpoint inhibitor responses.

BACKGROUND & AIMS: Tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) are the only two classes of FDA-approved drugs for individuals with advanced hepatocellular carcinoma (HCC). While TKIs confer only modest survival benefits, ICIs have been associated with remarkable outcomes but only in the minority of patients who respond. Understanding the mechanisms that determine the efficacy of ICIs in HCC will help to stratify patients likely to respond to ICIs. This study aims to elucidate how genetic composition and specific oncogenic pathways regulate the immune composition of HCC, which directly affects response to ICIs. METHODS: A collection of mouse HCCs with genotypes that closely simulate the genetic composition found in human HCCs were established using genome-editing approaches involving the delivery of transposon and CRISPR-Cas9 systems by hydrodynamic tail vein injection. Mouse HCC tumors were analyzed by RNA-sequencing while tumor-infiltrating T cells were analyzed by flow cytometry and single-cell RNA-sequencing. RESULTS: Based on the CD8+ T cell-infiltration level, we characterized tumors with different genotypes into cold and hot tumors. Anti-PD-1 treatment had no effect in cold tumors but was greatly effective in hot tumors. As proof-of-concept, a cold tumor (Trp53KO/MYCOE) and a hot tumor (Keap1KO/MYCOE) were further characterized. Tumor-infiltrating CD8+ T cells from Keap1KO/MYCOE HCCs expressed higher levels of proinflammatory chemokines and exhibited enrichment of a progenitor exhausted CD8+ T-cell phenotype compared to those in Trp53KO/MYCOE HCCs. The TKI sorafenib sensitized Trp53KO/MYCOE HCCs to anti-PD-1 treatment. CONCLUSION: Single anti-PD-1 treatment appears to be effective in HCCs with genetic mutations driving hot tumors, while combined anti-PD-1 and sorafenib treatment may be more appropriate in HCCs with genetic mutations driving cold tumors. IMPACT AND IMPLICATIONS: Genetic alterations of different driver genes in mouse liver cancers are associated with tumor-infiltrating CD8+ T cells and anti-PD-1 response. Mouse HCCs with different genetic compositions can be grouped into hot and cold tumors based on the level of tumor-infiltrating CD8+ T cells. This study provides proof-of-concept evidence to show that hot tumors are responsive to anti-PD-1 treatment while cold tumors are more suitable for combined treatment with anti-PD-1 and sorafenib. Our study might help to guide the design of patient stratification systems for single or combined treatments involving anti-PD-1.

Transitional premonocytes emerge in the periphery for host defense against bacterial infections.

Circulating Ly6Chi monocytes often undergo cellular death upon exhaustion of their antibacterial effector functions, which limits their capacity for subsequent macrophage differentiation. This shrouds the understanding on how the host replaces the tissue-resident macrophage niche effectively during bacterial invasion to avert infection morbidity. Here, we show that proliferating transitional premonocytes (TpMos), an immediate precursor of mature Ly6Chi monocytes (MatMos), were mobilized into the periphery in response to acute bacterial infection and sepsis. TpMos were less susceptible to apoptosis and served as the main source of macrophage replenishment when MatMos were vulnerable toward bacteria-induced cellular death. Furthermore, TpMo and its derived macrophages contributed to host defense by balancing the proinflammatory cytokine response of MatMos. Consequently, adoptive transfer of TpMos improved the survival outcome of lethal sepsis. Our findings hence highlight a protective role for TpMos during bacterial infections and their contribution toward monocyte-derived macrophage heterogeneity in distinct disease outcomes.

Hypoxia-induced macropinocytosis represents a metabolic route for liver cancer.

Hepatocellular carcinoma (HCC) invariably exhibits inadequate O2 (hypoxia) and nutrient supply. Hypoxia-inducible factor (HIF) mediates cascades of molecular events that enable cancer cells to adapt and propagate. Macropinocytosis is an endocytic process initiated by membrane ruffling, causing the engulfment of extracellular fluids (proteins), protein digestion and subsequent incorporation into the biomass. We show that macropinocytosis occurs universally in HCC under hypoxia. HIF-1 activates the transcription of a membrane ruffling protein, EH domain-containing protein 2 (EHD2), to initiate macropinocytosis. Knockout of HIF-1 or EHD2 represses hypoxia-induced macropinocytosis and prevents hypoxic HCC cells from scavenging protein that support cell growth. Germline or somatic deletion of Ehd2 suppresses macropinocytosis and HCC development in mice. Intriguingly, EHD2 is overexpressed in HCC. Consistently, HIF-1 or macropinocytosis inhibitor suppresses macropinocytosis and HCC development. Thus, we show that hypoxia induces macropinocytosis through the HIF/EHD2 pathway in HCC cells, harnessing extracellular protein as a nutrient to survive.

Inhibition of CMTM4 Sensitizes Cholangiocarcinoma and Hepatocellular Carcinoma to T Cell-Mediated Antitumor Immunity Through PD-L1.

Liver cancers consist primarily of hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC). Immune checkpoint inhibitors have emerged as promising therapeutic agents against liver cancers. Programmed cell death protein 1 (PD-1) is an immunoinhibitory receptor present on T cells that interacts with its ligand programmed death-ligand 1 (PD-L1) found on cancer cells. Blocking PD-1/PD-L1 binding improves T-cell survival, proliferation and cytotoxicity, which enhances their antitumor activity. Better understanding of the molecular mechanisms governing PD-1/PD-L1 response is essential to the development of predictive markers and therapeutic combinations that could improve the efficiency of anti-PD-1/PD-L1 treatment. Chemokine-like factor (CKLF)-like MARVEL transmembrane domain-containing 6 (CMTM6) has been recently identified as a major regulator of PD-L1. Another member in the CMTM family, CKLF-like MARVEL transmembrane domain-containing 4 (CMTM4), has been shown to compensate for the effects of CMTM6 when CMTM6 is lost. Interestingly, we found that CMTM4 is the major regulator of PD-L1 in the context of liver cancer. Up-regulated CMTM4 in patients with HCC and ICC is associated with poor patient survival, potentially due to its function in stabilizing PD-L1 expression, hence facilitating escape from T cell-mediated cytotoxicity. We confirmed the role of CMTM4 as a positive regulator of PD-L1 in multiple HCC and ICC cell lines and demonstrated that CMTM4 stabilizes PD-L1 through posttranslational mechanisms. In vivo, suppression of Cmtm4 inhibited HCC growth and increased CD8+ T-cell infiltration in immunocompetent mice. Furthermore, we found that depletion of CMTM4 sensitized HCC tumor to anti-PD-L1 treatment compared with control. This suggests that CMTM4 expression level could be a predictive marker for patient response to anti-PD-L1 treatment, and CMTM4 depletion can potentially be used to enhance the clinical benefits of anti-PD-L1 immunotherapy in patients with liver cancer.

Genome-wide CRISPR-Cas9 knockout library screening identified PTPMT1 in cardiolipin synthesis is crucial to survival in hypoxia in liver cancer.

Hypoxia, low oxygen (O2), is a key feature of all solid cancers, including hepatocellular carcinoma (HCC). Genome-wide CRISPR-Cas9 knockout library screening is used to identify reliable therapeutic targets responsible for hypoxic survival in HCC. We find that protein-tyrosine phosphatase mitochondrial 1 (PTPMT1), an important enzyme for cardiolipin (CL) synthesis, is the most significant gene and ranks just after hypoxia-inducible factor (HIF)-1α and HIF-1ß as crucial to hypoxic survival. CL constitutes the mitochondrial membrane and ensures the proper assembly of electron transport chain (ETC) complexes for efficient electron transfer in respiration. ETC becomes highly unstable during hypoxia. Knockout of PTPMT1 stops the maturation of CL and impairs the assembly of ETC complexes, leading to further electron leakage and ROS accumulation at ETC in hypoxia. Excitingly, HCC cells, especially under hypoxic conditions, show great sensitivity toward PTPMT1 inhibitor alexidine dihydrochloride (AD). This study unravels the protective roles of PTPMT1 in hypoxic survival and cancer development.

NIR-II-Excited Intravital Two-Photon Microscopy Distinguishes Deep Cerebral and Tumor Vasculatures with an Ultrabright NIR-I AIE Luminogen.

Intravital fluorescence imaging of vasculature morphology and dynamics in the brain and in tumors with large penetration depth and high signal-to-background ratio (SBR) is highly desirable for the study and theranostics of vascular-related diseases and cancers. Herein, a highly bright fluorophore (BTPETQ) with long-wavelength absorption and aggregation-induced near-infrared (NIR) emission (maximum at ≈700 nm) is designed for intravital two-photon fluorescence (2PF) imaging of a mouse brain and tumor vasculatures under NIR-II light (1200 nm) excitation. BTPETQ dots fabricated via nanoprecipitation show uniform size of around 42 nm and a high quantum yield of 19 ± 1% in aqueous media. The 2PF imaging of the mouse brain vasculatures labeled by BTPETQ dots reveals a 3D blood vessel network with an ultradeep depth of 924 µm. In addition, BTPETQ dots show enhanced 2PF in tumor vasculatures due to their unique leaky structures, which facilitates the differentiation of normal blood vessels from tumor vessels with high SBR in deep tumor tissues. Moreover, the extravasation and accumulation of BTPETQ dots in deep tumor (more than 900 µm) is visualized under NIR-II excitation. This study highlights the importance of developing NIR-II light excitable efficient NIR fluorophores for in vivo deep tissue and high contrast tumor imaging.

A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGFα-Mediated Recruitment of Neutrophils.

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor α (VEGF-α) by a minor subset of activated EpCAM+CD59+Ly-6D+ cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guérin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

Polymerization-Enhanced Two-Photon Photosensitization for Precise Photodynamic Therapy.

Two-photon excited photodynamic therapy (2PE-PDT) has attracted great attention in recent years due to its great potential for deep-tissue and highly spatiotemporally precise cancer therapy. Photosensitizers (PSs) with high singlet oxygen (1O2) generation efficiency and large two-photon absorption (2PA) cross-sections are highly desirable, but the availability of such PSs is limited by challenges in molecular design. In this work, we report that the polymerization of small-molecule PSs with aggregation-induced emission (AIE) could yield conjugated polymer PSs with good brightness, high 1O2 generation efficiency, and large 2PA cross-sections. A pair of conjugated polymer PSs were designed and synthesized, and the corresponding AIE PS dots were prepared by nanoprecipitation, which exhibited outstanding 2PE-PDT performance in in vitro cancer cell ablation and in vivo zebrafish liver tumor treatment. Our work highlights a strategy to design highly efficient PSs for 2PE-PDT.

Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen.

The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes.

Polymeric nanorods with aggregation-induced emission characteristics for enhanced cancer targeting and imaging.

Polymeric nanorods loaded with AIEgens are synthesized via nano-precipitation under ultrasound sonication, where prolonged sonication time could induce a nanodot-to-nanorod transition. These AIE nanorods, but not the nanodots, could be selectively internalized into cancer cells, which show better tumor accumulation, higher tumor penetration and more efficient in vivo cancer cell uptake.

Imaging of Inflammatory Responses in the Mouse Ear Skin.

The skin is one of the most physiologically important organs where the organism comes into contact with the external environment and is often a site where pathogen entry first occurs. Thus, a better understanding of the specialized cellular behavior of the immune system in the skin may be important for the improved treatment of diseases. Here, we describe in detail a procedure to image the dorsal mouse ear skin, using a customized ear stage and its associated coverslip holder, with an upright multiphoton microscope. As a demonstrative example, we describe the specific protocol for visualizing robust neutrophil trafficking in albino lysozyme-EGFP mice in response to zymosan particles. Instructive sections are provided for the mouse ear preparation, intradermal delivery of zymosan, design and use of the custom ear stage, as well as a solution for the uninterrupted live imaging of mice during prolonged sessions within a dark box. The mouse ear is easily accessible for imaging, and unlike most other organs, does not require any invasive surgery to be performed.

Illuminating the covert mission of mononuclear phagocytes in their regional niches.

Monocytes, dendritic cells (DCs) and macrophages have been classically categorized into the mononuclear phagocyte system (MPS) based on their similar functional and phenotypic characteristics. While an increasing amount of research has revealed substantial ontogenic and functional differences among these cells, the reasons behind their heterogeneity and strategic positioning in specific niches throughout the body are yet to be fully elucidated. In this review, we outline how recent advances in intravital imaging studies have dissected this phenomenon and have allowed us to appreciate how MPS cells exploit their regional niches to specialize and maximize their functional properties. Understanding their cellular behavior in each of their specialized microenvironment will eventually allow us to target specific cells and their behavioral patterns for improved vaccine and therapeutic purposes.

Induced-Pluripotent-Stem-Cell-Derived Primitive Macrophages Provide a Platform for Modeling Tissue-Resident Macrophage Differentiation and Function.

Tissue macrophages arise during embryogenesis from yolk-sac (YS) progenitors that give rise to primitive YS macrophages. Until recently, it has been impossible to isolate or derive sufficient numbers of YS-derived macrophages for further study, but data now suggest that induced pluripotent stem cells (iPSCs) can be driven to undergo a process reminiscent of YS-hematopoiesis in vitro. We asked whether iPSC-derived primitive macrophages (iMacs) can terminally differentiate into specialized macrophages with the help of growth factors and organ-specific cues. Co-culturing human or murine iMacs with iPSC-derived neurons promoted differentiation into microglia-like cells in vitro. Furthermore, murine iMacs differentiated in vivo into microglia after injection into the brain and into functional alveolar macrophages after engraftment in the lung. Finally, iPSCs from a patient with familial Mediterranean fever differentiated into iMacs with pro-inflammatory characteristics, mimicking the disease phenotype. Altogether, iMacs constitute a source of tissue-resident macrophage precursors that can be used for biological, pathophysiological, and therapeutic studies.

Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2.

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.

Biocompatible Green and Red Fluorescent Organic Dots with Remarkably Large Two-Photon Action Cross Sections for Targeted Cellular Imaging and Real-Time Intravital Blood Vascular Visualization.

Fluorescent organic dots are emerging as promising bioimaging reagents because of their high brightness, good photostability, excellent biocompatibility, and facile surface functionalization. Organic dots with large two-photon absorption (TPA) cross sections are highly desired for two-photon fluorescence microscopy. In this work, we report two biocompatible and photostable organic dots fabricated by encapsulating tetraphenylethene derivatives within DSPE-PEG matrix. The two organic dots show absorption maxima at 425 and 483 nm and emit green and red fluorescence at 560 and 645 nm, with high fluorescence quantum yields of 64% and 22%, respectively. Both organic dots exhibit excellent TPA property in the range of 800-960 nm, affording upon excitation at 820 nm remarkably large TPA cross sections of 1.2×10(6) and 2.5×10(6) GM on the basis of dot concentration. The bare fluorophores and their organic dots are biocompatible and have been used to stain living cells for one- and two-photon fluorescence bioimagings. The cRGD-modified organic dots can selectively target integrin αvß3 overexpressing breast cancer cells for targeted imaging. The organic dots are also applied for real-time two-photon fluorescence in vivo visualization of the blood vasculature of mouse ear, providing the spatiotemporal information about the whole blood vascular network. These results demonstrate that the present fluorescent organic dots are promising candidates for living cell and tissue imaging.

Neutrophil mobilization via plerixafor-mediated CXCR4 inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow.

Blood neutrophil homeostasis is essential for successful host defense against invading pathogens. Circulating neutrophil counts are positively regulated by CXCR2 signaling and negatively regulated by the CXCR4-CXCL12 axis. In particular, G-CSF, a known CXCR2 signaler, and plerixafor, a CXCR4 antagonist, have both been shown to correct neutropenia in human patients. G-CSF directly induces neutrophil mobilization from the bone marrow (BM) into the blood, but the mechanisms underlying plerixafor-induced neutrophilia remain poorly defined. Using a combination of intravital multiphoton microscopy, genetically modified mice and novel in vivo homing assays, we demonstrate that G-CSF and plerixafor work through distinct mechanisms. In contrast to G-CSF, CXCR4 inhibition via plerixafor does not result in neutrophil mobilization from the BM. Instead, plerixafor augments the frequency of circulating neutrophils through their release from the marginated pool present in the lung, while simultaneously preventing neutrophil return to the BM. Our study demonstrates for the first time that drastic changes in blood neutrophils can originate from alternative reservoirs other than the BM, while implicating a role for CXCR4-CXCL12 interactions in regulating lung neutrophil margination. Collectively, our data provides valuable insights into the fundamental regulation of neutrophil homeostasis, which may lead to the development of improved treatment regimens for neutropenic patients.