TGF-ß1 activates neutrophil signaling and gene expression but not migration.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-ß) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-ß on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-ß signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-ß1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-ß1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time- and dose-dependent manner. Additionally, TGF-ß1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B4 (LTB4), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-ß1 alone does not induce secretion of LTB4. RNA-sequencing revealed that TGF-ß1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M (OSM) and vascular endothelial growth factor A (VEGFA). These new insights into the role and impact of TGF-ß1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

TGF-ß1 activates neutrophil signaling and gene expression but not migration.

Tumor-associated neutrophils are found in many types of cancer and are often reported to contribute to negative outcomes. The presence of transforming growth factor-beta (TGF-ß) in the tumor microenvironment reportedly contributes to the skewing of neutrophils to a more pro-tumor phenotype. The effects of TGF-ß on neutrophil signaling and migration are, however, unclear. We sought to characterize TGF-ß signaling in both primary human neutrophils and the neutrophil-like cell line HL-60 and determine whether it directly induces neutrophil migration. We found that TGF-ß1 does not induce neutrophil chemotaxis in transwell or underagarose migration assays. TGF-ß1 does activate canonical signaling through SMAD3 and noncanonical signaling through ERK1/2 in neutrophils in a time-and dose-dependent manner. Additionally, TGF-ß1 present in the tumor-conditioned media (TCM) of invasive breast cancer cells results in SMAD3 activation. We discovered that TCM induces neutrophils to secrete leukotriene B 4 (LTB 4 ), which is a lipid mediator important for amplifying the range of neutrophil recruitment. However, TGF-ß1 alone does not induce secretion of LTB 4 . RNA-sequencing revealed that TGF-ß1 and TCM alter gene expression in HL-60 cells, including the mRNA levels of the pro-tumor oncostatin M ( OSM ) and vascular endothelial growth factor A ( VEGFA ). These new insights into the role and impact of TGF-ß1 on neutrophil signaling, migration, and gene expression have significant implications in the understanding of the changes in neutrophils that occur in the tumor microenvironment.

A network of Gαi signaling partners is revealed by proximity labeling proteomics analysis and includes PDZ-RhoGEF.

G protein­coupled receptors (GPCRs) that couple to the Gαi family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for Gαi in regulating the migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors. Here, we used an intact cell proximity­based labeling coupled to tandem mass tag (TMT)­based quantitative proteomics analysis to identify proteins that selectively interacted with the GTP-bound form of Gαi1. Multiple targets were identified and validated with a BioID2-tagged, constitutively active Gαi1 mutant, suggesting a network of interactions for activated GαI proteins in intact cells. We showed that active Gαi1, but not Gαi2, stimulated one candidate protein, PDZ-RhoGEF (PRG), despite more than 85% sequence identity between the G proteins. We also demonstrated in primary human neutrophils that active Gαi likely regulated the polarization of phosphorylated myosin light chain, a process critical for migration, through the activation of PRG. The identification and characterization of new targets directly or indirectly regulated by Gαi will aid in the investigation of the functional roles of Gαi-coupled GPCRs in multiple biological processes.

Fibrotic lung disease inhibits immune responses to staphylococcal pneumonia via impaired neutrophil and macrophage function.

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal disease characterized by collagen deposition within the lung interstitium. Bacterial infection is associated with increased morbidity and more rapid mortality in IPF patient populations, and pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) are commonly isolated from the lungs of hospitalized patients with IPF. Despite this, the effects of fibrotic lung injury on critical immune responses to infection remain unknown. In the present study, we show that, like humans with IPF, fibrotic mice infected with MRSA exhibit increased morbidity and mortality compared with uninfected fibrotic mice. We determine that fibrosis conferred a defect in MRSA clearance compared with nonfibrotic mice, resulting from blunted innate immune responses. We show that fibrosis inhibited neutrophil intracellular killing of MRSA through impaired neutrophil elastase release and oxidative radical production. Additionally, we demonstrate that lung macrophages from fibrotic mice have impaired phagocytosis of MRSA. Our study describes potentially novel impairments of antimicrobial responses upon pulmonary fibrosis development, and our findings suggest a possible mechanism for why patients with IPF are at greater risk of morbidity and mortality related to infection.

The Recruitment of Neutrophils to the Tumor Microenvironment Is Regulated by Multiple Mediators.

Neutrophils sense and migrate towards chemotactic factors released at sites of infection/inflammation and contain the affected area using a variety of effector mechanisms. Aside from these established immune defense functions, neutrophils are emerging as one of the key tumor-infiltrating immune cells that influence cancer progression and metastasis. Neutrophil recruitment to the tumor microenvironment (TME) is mediated by multiple mediators including cytokines, chemokines, lipids, and growth factors that are secreted from cancer cells and cancer-associated stromal cells. However, the molecular mechanisms that underlie the expression and secretion of the different mediators from cancer cells and how neutrophils integrate these signals to reach and invade tumors remain unclear. Here, we discuss the possible role of the epithelial to mesenchymal transition (EMT) program, which is a well-established promoter of malignant potential in cancer, in regulating the expression and secretion of these key mediators. We also summarize and review our current understanding of the machineries that potentially control the secretion of the mediators from cancer cells, including the exocytic trafficking pathways, secretory autophagy, and extracellular vesicle-mediated secretion. We further reflect on possible mechanisms by which different mediators collaborate by integrating their signaling network, and particularly focus on TGF-ß, a cytokine that is highly expressed in invasive tumors, and CXCR2 ligands, which are crucial neutrophil recruiting chemokines. Finally, we highlight gaps in the field and the need to expand current knowledge of the secretory machineries and cross-talks among mediators to develop novel neutrophil targeting strategies as effective therapeutic options in the treatment of cancer.

The principles of directed cell migration.

Cells have the ability to respond to various types of environmental cues, and in many cases these cues induce directed cell migration towards or away from these signals. How cells sense these cues and how they transmit that information to the cytoskeletal machinery governing cell translocation is one of the oldest and most challenging problems in biology. Chemotaxis, or migration towards diffusible chemical cues, has been studied for more than a century, but information is just now beginning to emerge about how cells respond to other cues, such as substrate-associated cues during haptotaxis (chemical cues on the surface), durotaxis (mechanical substrate compliance) and topotaxis (geometric features of substrate). Here we propose four common principles, or pillars, that underlie all forms of directed migration. First, a signal must be generated, a process that in physiological environments is much more nuanced than early studies suggested. Second, the signal must be sensed, sometimes by cell surface receptors, but also in ways that are not entirely clear, such as in the case of mechanical cues. Third, the signal has to be transmitted from the sensing modules to the machinery that executes the actual movement, a step that often requires amplification. Fourth, the signal has to be converted into the application of asymmetric force relative to the substrate, which involves mostly the cytoskeleton, but perhaps other players as well. Use of these four pillars has allowed us to compare some of the similarities between different types of directed migration, but also to highlight the remarkable diversity in the mechanisms that cells use to respond to different cues provided by their environment.

Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-ß and CXCR2 Ligands.

Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-ß cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-ß, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-ß and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.

Getting TANned: How the tumor microenvironment drives neutrophil recruitment.

The directed migration of neutrophils to sites of injury or infection is mediated by complex networks of chemoattractant-receptor signaling cascades. The recent appreciation of neutrophils as active participants in tumor progression and metastasis has drawn attention to a number of chemokine-receptor systems that may drive their recruitment to tumors. However, the dynamic nature of the tumor microenvironment (TME) along with the phenotypic diversity among tumor-associated neutrophils (TANs) call for a more comprehensive approach to understand neutrophil trafficking to tumors. Here, we review recent advances in understanding how guidance cues underlie neutrophil migration to primary and secondary tumor sites. We also discuss how the presence of other myeloid cells, such as functionally diverse subsets of tumor-associated macrophages (TAMs), can further influence neutrophil accumulation in tumors. Finally, we highlight the importance of hypoxia sensing in localizing TAMs and TANs in the tumor niche and provide a cohesive view on how both myeloid cell types shape TME-associated extracellular matrix organization, which in turn contribute to tumor progression.

A Pseudomonas aeruginosa hepta-acylated lipid A variant associated with cystic fibrosis selectively activates human neutrophils.

Pseudomonas aeruginosa (PA) infection in cystic fibrosis (CF) lung disease causes airway neutrophilia and hyperinflammation without effective bacterial clearance. We evaluated the immunostimulatory activities of lipid A, the membrane anchor of LPS, isolated from mutants of PA that synthesize structural variants, present in the airways of patients with CF, to determine if they correlate with disease severity and progression. In a subset of patients with a severe late stage of CF disease, a unique hepta-acylated lipid A, hepta-1855, is synthesized. In primary human cell cultures, we found that hepta-1855 functioned as a potent TLR4 agonist by priming neutrophil respiratory burst and stimulating strong IL-8 from monocytes and neutrophils. hepta-1855 also had a potent survival effect on neutrophils. However, it was less efficient in stimulating neutrophil granule exocytosis and also less potent in triggering proinflammatory TNF-α response from monocytes. In PA isolates that do not synthesize hepta-1855, a distinct CF-specific adaptation favors synthesis of a penta-1447 and hexa-1685 LPS mixture. We found that penta-1447 lacked immunostimulatory activity but interfered with inflammatory IL-8 synthesis in response to hexa-1685. Together, these observations suggest a potential contribution of hepta-1855 to maintenance of the inflammatory burden in late-stage CF by recruiting neutrophils via IL-8 and promoting their survival, an effect presumably amplified by the absence of penta-1447. Moreover, the relative inefficiency of hepta-1855 in triggering neutrophil degranulation may partly explain the persistence of PA in CF disease, despite extensive airway neutrophilia.

Type I interferon signaling contributes to the bias that Toll-like receptor 4 exhibits for signaling mediated by the adaptor protein TRIF.

Signaling by Toll-like receptor 4 (TLR4) is mediated by either of two adaptor proteins: myeloid differentiation marker 88 (MyD88) or Toll-interleukin-1 (IL-1) receptor (TIR) domain-containing adaptor inducing interferon-ß (TRIF). Whereas MyD88-mediated signaling leads to proinflammatory responses, TRIF-mediated signaling leads to less toxic immunostimulatory responses that are beneficial in boosting vaccine responses. The hypothesis that monophosphorylated lipid A structures act as TRIF-biased agonists of TLR4 offered a potential mechanism to explain their clinical value as vaccine adjuvants, but studies of TRIF-biased agonists have been contradictory. In experiments with mouse dendritic cells, we found that irrespective of the agonist used, TLR4 functioned as a TRIF-biased signaling system through a mechanism that depended on the autocrine and paracrine effects of type I interferons. The TLR4 agonist synthetic lipid A induced expression of TRIF-dependent genes at lower concentrations than were necessary to induce the expression of genes that depend on MyD88-mediated signaling. Blockade of type I interferon signaling selectively decreased the potency of lipid A (increased the concentration required) in inducing the expression of TRIF-dependent genes, thereby eliminating adaptor bias. These data may explain how high-potency TLR4 agonists can act as clinically useful vaccine adjuvants by selectively activating TRIF-dependent signaling events required for immunostimulation, without or only weakly activating potentially harmful MyD88-dependent inflammatory responses.