Alyssa D. Gregory

Tumor-Associated Neutrophils: New Targets for Cancer Therapy

Studies have begun to emerge showing critical roles for neutrophils in tumorigenesis. Neutrophils can have a significant impact on the tumor microenvironment via their production of cytokines and chemokines, which influence inflammatory cell recruitment and activation. Additionally, products secreted from neutrophils, such as reactive oxygen species and proteinases, have defined and specific roles in regulating tumor cell proliferation, angiogenesis, and metastasis. Although evidence suggests that neutrophils act in a decidedly protumor capacity in vivo, recent studies indicate that neutrophils may be manipulated to exhibit cytotoxicity against tumors. Herein, we explore the idea of targeting tumor-associated neutrophils as a means of antitumor therapy and the important ramifications such manipulation could pose to host tissues.

G-CSF Is an Essential Regulator of Neutrophil Trafficking from the Bone Marrow to the Blood

Neutrophils are released from the bone marrow in a regulated fashion to maintain homeostatic levels in the blood and to respond to physiological stresses, including infection. We show that under basal conditions granulocyte colony-stimulating factor (G-CSF) is an essential regulator of neutrophil release from the bone marrow. Nonredundant signals generated by the membrane-proximal 87 amino acids of the G-CSF receptor (G-CSFR) are sufficient to mediate this response. Surprisingly, G-CSFR expression on neutrophils is neither necessary nor sufficient for their mobilization from the bone marrow, suggesting that G-CSF induces neutrophil mobilization indirectly through the generation of trans-acting signals. Evidence is provided suggesting that downregulation of stromal cell-derived factor 1 expression in the bone marrow may represent such a signal.

Neutrophil elastase–mediated degradation of IRS-1 accelerates lung tumor growth

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that tumor-associated inflammatory cells may modify lung tumor growth and invasiveness. To determine the role of neutrophil elastase (encoded by Elane) on tumor progression, we used the loxP-Stop-loxP K-rasG12D (LSL–K-ras) model of mouse lung adenocarcinoma to generate LSL–K-ras-Elane−/− mice. Tumor burden was markedly reduced in LSL–K-ras-Elane−/− mice at all time points after induction of mutant K-ras expression. Kaplan-Meier survival analysis showed that whereas all LSL–K-ras-Elane+/+ mice died, none of the mice lacking neutrophil elastase died. Neutrophil elastase directly induced tumor cell proliferation in both human and mouse lung adenocarcinomas by gaining access to an endosomal compartment within tumor cells, where it degraded insulin receptor substrate-1 (IRS-1). Immunoprecipitation studies showed that, as neutrophil elastase degraded IRS-1, there was increased interaction between phosphatidylinositol 3-kinase (PI3K) and the potent mitogen platelet-derived growth factor receptor (PDGFR), thereby skewing the PI3K axis toward tumor cell proliferation. The inverse relationship identified between neutrophil elastase and IRS-1 in LSL–K-ras mice was also identified in human lung adenocarcinomas, thus translating these findings to human disease. This study identifies IRS-1 as a key regulator of PI3K within malignant cells. Additionally, to our knowledge, this is the first description of a secreted proteinase gaining access to the inside of a cell and altering intracellular signaling.

Recruitment of the inflammatory subset of monocytes to sites of ischemia induces angiogenesis in a monocyte chemoattractant protein-1-dependent fashion

There is accumulating evidence that delivery of bone marrow cells to sites of ischemia by direct local injection or mobilization into the blood can stimulate angiogenesis. This has stimulated tremendous interest in the translational potential of angiogenic cell population(s) in the bone marrow to mediate therapeutic angiogenesis. However, the mechanisms by which these cells stimulate angiogenesis are unclear. Herein, we show that the inflammatory subset of monocytes is selectively mobilized into blood after surgical induction of hindlimb ischemia in mice and is selectively recruited to ischemic muscle. Adoptive‐transfer studies show that delivery of a small number of inflammatory monocytes early (within 48 h) of induction of ischemia results in a marked increase in the local production of MCP‐1, which in turn, is associated with a secondary, more robust wave of monocyte recruitment. Studies of mice genetically deficient in MCP‐1 or CCR2 indicate that although not required for the early recruitment of monocytes, the secondary wave of monocyte recruitment and subsequent stimulation of angiogenesis are dependent on CCR2 signaling. Collectively, these data suggest a novel role for MCP‐1 in the inflammatory, angiogenic response to ischemia.

Clathrin Pit-mediated Endocytosis of Neutrophil Elastase and Cathepsin G by Cancer Cells

Background: Neutrophil elastase (NE)-induced tumor cell proliferation requires endosomal entry of the proteinase. Results: NE and CG bind to the surface of cancer cells and enter into tumor endosomes in a clathrin- and dynamin-dependent, caveolin- and flotillin-independent fashion. Conclusion: Entry of NE and CG into tumor endosomes occurs via classic clathrin-pit mediated endocytosis, which requires the binding of the proteinase to a cell surface receptor. Significance: This novel means of cell entry may grant tumor cells access to numerous, as yet unidentified bioactive molecules located in the extracellular environment. Neutrophil elastase (NE) is a neutrophil-derived serine proteinase with broad substrate specificity. We have recently demonstrated that NE is capable of entering tumor cell endosomes and processing novel intracellular substrates. In the current study, we sought to determine the mechanism by which NE enters tumor cells. Our results show that NE enters into early endosomal antigen-1+ endosomes in a dynamin- and clathrin-dependent but flotillin-1- and caveolin-1-independent fashion. Cathepsin G (but not proteinase-3) also enters tumor endosomes via the same mechanism. We utilized 125I-labeled NE to demonstrate that NE binds to the surface of cancer cells. Incubation of radiolabeled NE with lung cancer cells displays a dissociation constant (Kd) of 284 nm. Because NE is known to bind to heparan sulfate- and chondroitin sulfate-containing proteoglycans, we treated cells with glycanases to remove these confounding factors, which did not significantly diminish cell surface binding or endosomal entry. Thus, NE and CG bind to the surface of cancer cells, presumably to a cell surface receptor, and subsequently undergo clathrin pit-mediated endocytosis.

Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE−/− mice are protected from asbestos‐induced lung fibrosis. NE−/− mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF‐β‐independent fashion. Treatment of asbestos‐recipient mice with ONO‐5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE‐inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.

Effects of intra-abdominal sepsis on atherosclerosis in mice.

IntroductionSepsis and other infections are associated with late cardiovascular events. Although persistent inflammation is implicated, a causal relationship has not been established. We tested whether sepsis causes vascular inflammation and accelerates atherosclerosis.MethodsWe performed prospective, randomized animal studies at a university research laboratory involving adult male ApoE-deficient (ApoE−/−) and young C57B/L6 wild-type (WT) mice. In the primary study conducted to determine whether sepsis accelerates atherosclerosis, we fed ApoE−/− mice (N = 46) an atherogenic diet for 4 months and then performed cecal ligation and puncture (CLP), followed by antibiotic therapy and fluid resuscitation or a sham operation. We followed mice for up to an additional 5 months and assessed atheroma in the descending aorta and root of the aorta. We also exposed 32 young WT mice to CLP or sham operation and followed them for 5 days to determine the effects of sepsis on vascular inflammation.ResultsApoE−/− mice that underwent CLP had reduced activity during the first 14 days (38% reduction compared to sham; P < 0.001) and sustained weight loss compared to the sham-operated mice (-6% versus +9% change in weight after CLP or sham surgery to 5 months; P < 0.001). Despite their weight loss, CLP mice had increased atheroma (46% by 3 months and 41% increase in aortic surface area by 5 months; P = 0.03 and P = 0.004, respectively) with increased macrophage infiltration into atheroma as assessed by immunofluorescence microscopy (0.52 relative fluorescence units (rfu) versus 0.97 rfu; P = 0.04). At 5 months, peritoneal cultures were negative; however, CLP mice had elevated serum levels of interleukin 6 (IL-6) and IL-10 (each at P < 0.05). WT mice that underwent CLP had increased expression of intercellular adhesion molecule 1 in the aortic lumen versus sham at 24 hours (P = 0.01) that persisted at 120 hours (P = 0.006). Inflammatory and adhesion genes (tumor necrosis factor α, chemokine (C-C motif) ligand 2 and vascular cell adhesion molecule 1) and the adhesion assay, a functional measure of endothelial activation, were elevated at 72 hours and 120 hours in mice that underwent CLP versus sham-operations (all at P <0.05).ConclusionsUsing a combination of existing murine models for atherosclerosis and sepsis, we found that CLP, a model of intra-abdominal sepsis, accelerates atheroma development. Accelerated atheroma burden was associated with prolonged systemic, endothelial and intimal inflammation and was not explained by ongoing infection. These findings support observations in humans and demonstrate the feasibility of a long-term follow-up murine model of sepsis.

Systemic levels of G‐CSF and interleukin‐6 determine the angiogenic potential of bone marrow resident monocytes

There is considerable interest in the potential of cell‐based approaches to mediate therapeutic angiogenesis for acute and chronic vascular syndromes. Using a mouse model of HLI, we showed previously that adoptive transfer of a small number of donor monocytes enhanced revascularization significantly. Herein, we provide data suggesting that the BM resident monocytes sense systemic signals that influence their future functional capacity. Specifically, following induction of distant ischemia, the angiogenic capacity of BM resident monocytes is reduced markedly. We provide evidence that G‐CSF and IL‐6 represent such “conditioning” signals. Systemic levels of G‐CSF and IL‐6 are increased significantly following induction of HLI. Accordingly, BM resident monocytes from ischemic mice exhibited increased pSTAT3 and STAT3 target gene expression. Finally, G‐CSFR−/− and IL‐6−/− mice were resistant to the deleterious effects of ischemic conditioning on monocyte angiogenic potential. RNA expression profiling suggested that ischemia‐conditioned monocytes in the BM up‐regulate the well‐described M2 polarization markers Chi3l4 and Lrg1. Consistent with this observation, M2‐skewed monocytes from SHIP−/− mice also had impaired angiogenic capacity. Collectively, these data show that G‐CSF and IL‐6 provide signals that determine the angiogenic potential of BM resident monocytes.

Thrombospondin-1 restrains neutrophil granule serine protease function and regulates the innate immune response during Klebsiella pneumoniae infection

Neutrophil elastase (NE) and cathepsin G (CG) contribute to intracellular microbial killing but, if left unchecked and released extracellularly, promote tissue damage. Conversely, mechanisms that constrain neutrophil serine protease activity protect against tissue damage but may have the untoward effect of disabling the microbial killing arsenal. The host elaborates thrombospondin-1 (TSP-1), a matricellular protein released during inflammation, but its role during neutrophil activation following microbial pathogen challenge remains uncertain. Mice deficient in TSP-1 (thbs1−/−) showed enhanced lung bacterial clearance, reduced splenic dissemination, and increased survival compared with wild-type (WT) controls during intrapulmonary Klebsiella pneumoniae infection. More effective pathogen containment was associated with reduced burden of inflammation in thbs1−/− mouse lungs compared with WT controls. Lung NE activity was increased in thbs1−/− mice following K. pneumoniae challenge, and thbs1−/− neutrophils showed enhanced intracellular microbial killing that was abrogated with recombinant TSP-1 administration or WT serum. Thbs1−/− neutrophils exhibited enhanced NE and CG enzymatic activity, and a peptide corresponding to amino-acid residues 793–801 within the type-III repeat domain of TSP-1 bridled neutrophil proteolytic function and microbial killing in vitro. Thus, TSP-1 restrains proteolytic action during neutrophilic inflammation elicited by K. pneumoniae, providing a mechanism that may regulate the microbial killing arsenal.

Mouse Genome-Wide Association Study of Preclinical Group II Pulmonary Hypertension Identifies Epidermal Growth Factor Receptor

&NA; Pulmonary hypertension (PH) is associated with features of obesity and metabolic syndrome that translate to the induction of PH by chronic high‐fat diet (HFD) in some inbred mouse strains. We conducted a genome‐wide association study (GWAS) to identify candidate genes associated with susceptibility to HFD‐induced PH. Mice from 36 inbred and wild‐derived strains were fed with regular diet or HFD for 20 weeks beginning at 6‐12 weeks of age, after which right ventricular (RV) and left ventricular (LV) end‐systolic pressure (ESP) and maximum pressure (MaxP) were measured by cardiac catheterization. We tested for association of RV MaxP and RV ESP and identified genomic regions enriched with nominal associations to both of these phenotypes. We excluded genomic regions if they were also associated with LV MaxP, LV ESP, or body weight. Genes within significant regions were scored based on the shortest‐path betweenness centrality, a measure of network connectivity, of their human orthologs in a gene interaction network of human PH‐related genes. WSB/EiJ, NON/ShiLtJ, and AKR/J mice had the largest increases in RV MaxP after high‐fat feeding. Network‐based scoring of GWAS candidates identified epidermal growth factor receptor (Egfr) as having the highest shortest‐path betweenness centrality of GWAS candidates. Expression studies of lung homogenate showed that EGFR expression is increased in the AKR/J strain, which developed a significant increase in RV MaxP after high‐fat feeding as compared with C57BL/6J, which did not. Our combined GWAS and network‐based approach adds evidence for a role for Egfr in murine PH.