Julie Laval

Activation of critical, host-induced, metabolic and stress pathways marks neutrophil entry into cystic fibrosis lungs

Cystic fibrosis (CF) patients undergo progressive airway destruction caused in part by chronic neutrophilic inflammation. While opportunistic pathogens infecting CF airways can cause inflammation, we hypothesized that host-derived metabolic and stress signals would also play a role in this process. We show that neutrophils that have entered CF airways have increased phosphorylation of the eukaryotic initiation factor 4E and its partner the 4E-binding protein 1; 2 key effectors in the growth factor- and amino acid-regulated mammalian target of rapamycin (mTOR) pathway. Furthermore CF airway neutrophils display increased phosphorylation of the cAMP response element binding protein (CREB), a major transcriptional coactivator in stress signaling cascades. These active intracellular pathways are associated with increased surface expression of critical adaptor molecules, including the growth factor receptor CD114 and the receptor for advanced glycation end-products (RAGE), a CREB inducer and sensor for host-derived damage-associated molecular patterns (DAMPs). Most CF airway fluids lack any detectable soluble RAGE, an inhibitory decoy receptor for DAMPs. Concomitantly, CF airway fluids displayed high and consequently unopposed levels of S100A12; a potent mucosa- and neutrophil-derived DAMP. CF airway neutrophils also show increased surface levels of 2 critical CREB targets, the purine-recycling enzyme CD39 and the multifunctional, mTOR-inducing CXCR4 receptor. This coordinated set of events occurs in all patients, even in the context of minimal airway inflammation and well-preserved lung function. Taken together, our data demonstrate an early and sustained activation of host-responsive metabolic and stress pathways upon neutrophil entry into CF airways, suggesting potential targets for therapeutic modulation.

Metabolic Adaptation of Neutrophils in Cystic Fibrosis Airways Involves Distinct Shifts in Nutrient Transporter Expression

Inflammatory conditions can profoundly alter human neutrophils, a leukocyte subset generally viewed as terminally differentiated and catabolic. In cystic fibrosis (CF) patients, neutrophils recruited to CF airways show active exocytosis and sustained phosphorylation of prosurvival, metabolic pathways. Because the CF airway lumen is also characterized by high levels of free glucose and amino acids, we compared surface expression of Glut1 (glucose) and ASCT2 (neutral amino acids) transporters, as well as that of PiT1 and PiT2 (inorganic phosphate transporters), in blood and airway neutrophils, using specific retroviral envelope-derived ligands. Neither nutrient transporter expression nor glucose uptake was altered on blood neutrophils from CF patients compared with healthy controls. Notably, however, airway neutrophils of CF patients had higher levels of PiT1 and Glut1 and increased glucose uptake compared with their blood counterparts. Based on primary granule exocytosis and scatter profiles, CF airway neutrophils could be divided into two subsets, with one of the subsets characterized by more salient increases in Glut1, ASCT2, PiT1, and PiT2 expression. Moreover, in vitro exocytosis assays of blood neutrophils suggest that surface nutrient transporter expression is not directly associated with primary (or secondary) granule exocytosis. Although expression of nutrient transporters on CF blood or airway neutrophils was not altered by genotype, age, gender, or Pseudomonas aeruginosa infection, oral steroid treatment decreased Glut1 and PiT2 levels in blood neutrophils. Thus, neutrophils recruited from blood into the CF airway lumen display augmented cell surface nutrient transporter expression and glucose uptake, consistent with metabolic adaptation.

Optimization of tumor xenograft dissociation for the profiling of cell surface markers and nutrient transporters

Metabolic adaptations and changes in the expression of nutrient transporters are known to accompany tumorigenic processes. Nevertheless, in the context of solid tumors, studies of metabolism are hindered by a paucity of tools allowing the identification of cell surface transporters on individual cells. Here, we developed a method for the dissociation of human breast cancer tumor xenografts combined with quantification of cell surface markers, including metabolite transporters. The expression profiles of four relevant nutrient transporters for cancer cells’ metabolism, Glut1, ASCT2, PiT1 and PiT2 (participating to glucose, glutamine and inorganic phosphate, respectively), as detected by new retroviral envelope glycoprotein-derived ligands, were distinctive of each tumor, unveiling underlying differences in metabolic pathways. Our tumor dissociation procedure and nutrient transporter profiling technology provides opportunities for future basic research, clinical diagnosis, prognosis and evaluation of therapeutic responses, as well as for drug discovery and development.

Neutrophils in cystic fibrosis.

Abstract Cystic fibrosis (CF) lung disease is characterized by chronic infection and inflammation. Among inflammatory cells, neutrophils represent the major cell population accumulating in the airways of CF patients. While neutrophils provide the first defensive cellular shield against bacterial and fungal pathogens, in chronic disease conditions such as CF these short-lived immune cells release their toxic granule contents that cause tissue remodeling and irreversible structural damage to the host. A variety of human and murine studies have analyzed neutrophils and their products in the context of CF, yet their precise functional role and therapeutic potential remain controversial and incompletely understood. Here, we summarize the current evidence in this field to shed light on the complex and multi-faceted role of neutrophils in CF lung disease.

Abstract B142: Dissociation of preclinical primary human cancer xenografts for cell surface transportome profiling.

Background: Tumor cell metabolism is of growing interest in both basic and clinical cancer research. A better understanding of underlying molecular mechanisms involving metabolite transport in normal and tumor cells should help drug discovery and development. Specific exofacial ligands to metabolite transporters derived from the receptor binding domain (RBD) of retrovirus envelope proteins were developed and used to quantify cell surface metabolite transporters. While cell surface labelling can be readily performed on cultured cell lines, analysis of single cells from solid tumors is more challenging. In this study, we developed a robust method for the disaggregation of tumor cells from human breast cancers grown as xenografts in mice. This procedure was then used to analyse the expression profiles of 4 cell membrane metabolite transporters involved in cell proliferation and tumorigenesis: Glut1, ASCT2, PiT1, and PiT2. Materials and methods: Eight primary human breast cancer xenografts were used for ex vivo experiments (Marangoni et al 2007). We developed an optimized disaggregation protocol to obtain maximum viable cell recovery from the xenografts. The protocol was validated for presence of CD44+ tumor cells and for cell viability using caspase 3 and DAPI exclusion and subsequently, applied to the xenografts for flow cytometry analyses, immunohistochemistry and ex vivo cell culture. Expression profiles of 4 metabolite transporters were assessed in 5 different human breast cancer xenografts. Results: The optimal dissociation protocol developed for these tumors combined mild non-enzymatic (non-enzymatic dissociation buffer) and enzymatic (collagenase III/DNase I) steps, followed by cell purification on a dual density Ficoll gradient. Less than 10% of resulting DAPI negative tumor cells were caspase 3 positive. Dissociated cells showed sustained viability in in vitro cultures for at least 12 days. The numbers of CD44+ cells determined by flow cytometry corresponded to those observed by IHC. The expression profiles of Glut1, ASCT2, PiT1, and PiT2 were distinct for each of the five human breast cancers, and metabolite transporter profiles were highly conserved for xenografts derived from the same tumor. Conclusions: Mouse xenograft implants of human breast cancer tumors were used to optimize and validate a dissociation method for the production of viable single cells. Cell suspensions were then assessed for cell surface metabolite transporters expression by flow cytometry. The expression patterns of four metabolite transporters, Glut1, ASCT2, PiT1, and PiT2 showed distinctive signature profiles for each group of xenografts, indicative of specific metabolic adaptations that can be tracked with our ligands for each tumor. Reference: 1. Marangoni E et al, CCR 2007;13:3989–3998. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B142.

Innate Immunity of the Lung: From Basic Mechanisms to Translational Medicine

The respiratory tract is faced daily with 10,000 L of inhaled air. While the majority of air contains harmless environmental components, the pulmonary immune system also has to cope with harmful microbial or sterile threats and react rapidly to protect the host at this intimate barrier zone. The airways are endowed with a broad armamentarium of cellular and humoral host defense mechanisms, most of which belong to the innate arm of the immune system. The complex interplay between resident and infiltrating immune cells and secreted innate immune proteins shapes the outcome of host-pathogen, host-allergen, and host-particle interactions within the mucosal airway compartment. Here, we summarize and discuss recent findings on pulmonary innate immunity and highlight key pathways relevant for biomarker and therapeutic targeting strategies for acute and chronic diseases of the respiratory tract.

Aktuelle Ansätze und Kontroversen zu angeborener Immunität und Lungenerkrankung bei zystischer Fibrose

Die Lungenerkrankung bei zystischer Fibrose (CF) ist gekennzeichnet durch chronische Infektion und Inflammation. Bei der entzündlichen Reaktion im Rahmen der CF steht die Aktivierung des angeborenen Immunsystems im Vordergrund. Bakterien und Pilze sind die wichtigsten Krankheitserreger, die bei CF chronisch die Atemwege besiedeln. In Reaktion auf die mikrobiologische Bedrohung, die von speziellen Rezeptoren des angeborenen Immunsystems auf Atemwegsepithelzellen und myeloischen Zellen erkannt wird, werden chemische Lockstoffe ausgeschüttet, die neutrophile Granulozyten in großer Zahl in die von CF betroffenen Atemwege rekrutieren. Den Neutrophilen gelingt es jedoch nicht, die invasiven Krankheitserreger wirksam zu eliminieren; stattdessen setzen sie schädigende Proteasen und Oxidanzien frei und verursachen letztlich Gewebeschäden. Mit diesem Beitrag liefern wir eine Zusammenfassung und Übersicht zu aktuellen Ansätzen und Kontroversen zum Thema der angeborenen Immunität bei CF-Lungenerkrankung und gehen dabei insbesondere auf die ungeklärten Fragen ein, ob die Inflammation bei CF positiv oder negativ ist und wie die Mechanismen der angeborenen Immunität therapeutisch genutzt werden könnten. Übersetzung aus J Innate Immun 2016;8:531-540 (DOI: 10.1159/000446840)