Modulation of neutrophil degranulation by hypoxia

Neutrophils are key effector cells of the innate immune system. They employ a number of powerful ‘weapons’ to eliminate pathogens, including an array of destructive proteins packaged into distinctive granule subsets. In addition to their microbicidal activity, these granule proteins are capable of causing substantial tissue damage if inappropriately deployed. To mitigate against this possibility, most physiological stimuli induce minimal extracellular degranulation. Sites of inflammation and infection are usually hypoxic, and it has been shown that oxygen depletion compromises neutrophil function by impairing the generation of reactive oxygen species and hence bacterial killing. The key finding reported in this thesis is that hypoxia substantially increases the release of all neutrophil granule subsets, as measured by the release of (active) hallmark proteins (elastase, myeloperoxidase, lactoferrin and matrix metalloproteinase-9). In consequence, supernatants from hypoxic neutrophils induced substantially more damage to lung epithelial cell layers than supernatants from neutrophils cultured under normoxic conditions; this damage was protein- and protease-dependent. This pattern of damage was seen consistently across lung adenocarcinoma-derived epithelial cells, primary immortalised lung epithelial cells, and primary human bronchial epithelial cells grown in physiological air-liquid interface culture. Surprisingly, the mechanism of hypoxia-augmented degranulation was found to be independent of protein synthesis and specifically, of the transcription factor HIF-1α (the ‘master-regulator’ of hypoxic responses); thus, hypoxia did not affect mRNA transcript or protein abundance of the major granule components, and hypoxia mimetics failed to recapitulate the phenotype. Inhibition of the key pathways known to be involved in neutrophil degranulation, including, phosphatidylinositol 3-kinase and phospholipase C, but not calcium flux prevented augmented granule release under hypoxia In conclusion, hypoxia induces a destructive neutrophil phenotype, with increased release of multiple histotoxic proteases. This may contribute to tissue injury and disease pathogenesis in a range of clinically important conditions.This work was supported by the British Lung Foundatio

Phenotype of ARDS alveolar and blood neutrophils

RATIONALE: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied. OBJECTIVES: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition. METHODS: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays. MEASUREMENTS AND MAIN RESULTS: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures. CONCLUSIONS: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.This work was funded by a non-commercial grant from GSK, with additional support from The Wellcome Trust, Papworth Hospital, The British Lung Foundation and the NIHR Cambridge Biomedical Research Centre. DMLS holds a Gates Cambridge Scholarship; CS is in receipt of a Wellcome Trust Early Postdoctoral Research Fellowship for Clinician Scientists [WT101692MA].This is the author accepted manuscript. The final version is available from ATS Journals via http://dx.doi.org/10.1164/rccm.201509-1818O

Circulating BMP9 Protects the Pulmonary Endothelium during Inflammation-induced Lung Injury in Mice.

Rationale: Pulmonary endothelial permeability contributes to the high-permeability pulmonary edema that characterizes acute respiratory distress syndrome. Circulating BMP9 (bone morphogenetic protein 9) is emerging as an important regulator of pulmonary vascular homeostasis. Objectives:To determine whether endogenous BMP9 plays a role in preserving pulmonary endothelial integrity and whether loss of endogenous BMP9 occurs during LPS challenge. Methods: A BMP9-neutralizing antibody was administrated to healthy adult mice, and lung vasculature was examined. Potential mechanisms were delineated by transcript analysis in human lung endothelial cells. The impact of BMP9 administration was evaluated in a murine acute lung injury model induced by inhaled LPS. Levels of BMP9 were measured in plasma from patients with sepsis and from endotoxemic mice. Measurements and Main Results: Subacute neutralization of endogenous BMP9 in mice (N = 12) resulted in increased lung vascular permeability (P = 0.022), interstitial edema (P = 0.0047), and neutrophil extravasation (P = 0.029) compared with IgG control treatment (N = 6). In pulmonary endothelial cells, BMP9 regulated transcriptome pathways implicated in vascular permeability and cell-membrane integrity. Augmentation of BMP9 signaling in mice (N = 8) prevented inhaled LPS-induced lung injury (P = 0.0027) and edema (P < 0.0001). In endotoxemic mice (N = 12), endogenous circulating BMP9 concentrations were markedly reduced, the causes of which include a transient reduction in hepatic BMP9 mRNA expression and increased elastase activity in plasma. In human patients with sepsis (N = 10), circulating concentratons of BMP9 were also markedly reduced (P < 0.0001). Conclusions: Endogenous circulating BMP9 is a pulmonary endothelial-protective factor, downregulated during inflammation. Exogenous BMP9 offers a potential therapy to prevent increased pulmonary endothelial permeability in lung injury

NBEAL2 is required for neutrophil and NK cell function and pathogen defense.

Mutations in the human NBEAL2 gene cause gray platelet syndrome (GPS), a bleeding diathesis characterized by a lack of α granules in platelets. The functions of the NBEAL2 protein have not been explored outside platelet biology, but there are reports of increased frequency of infection and abnormal neutrophil morphology in patients with GPS. We therefore investigated the role of NBEAL2 in immunity by analyzing the phenotype of Nbeal2-deficient mice. We found profound abnormalities in the Nbeal2-deficient immune system, particularly in the function of neutrophils and NK cells. Phenotyping of Nbeal2-deficient neutrophils showed a severe reduction in granule contents across all granule subsets. Despite this, Nbeal2-deficient neutrophils had an enhanced phagocyte respiratory burst relative to Nbeal2-expressing neutrophils. This respiratory burst was associated with increased expression of cytosolic components of the NADPH oxidase complex. Nbeal2-deficient NK cells were also dysfunctional and showed reduced degranulation. These abnormalities were associated with increased susceptibility to both bacterial (Staphylococcus aureus) and viral (murine CMV) infection in vivo. These results define an essential role for NBEAL2 in mammalian immunity

Eros is a novel transmembrane protein that controls the phagocyte respiratory burst and is essential for innate immunity

The phagocyte respiratory burst is crucial for innate immunity. The transfer of electrons to oxygen is mediated by a membrane-bound heterodimer, comprising gp91$\textit{phox}$ and p22$\textit{phox}$ subunits. Deficiency of either subunit leads to severe immunodeficiency. We describe Eros (essential for reactive oxygen species), a protein encoded by the previously undefined mouse gene $\textit{bc017643}$, and show that it is essential for host defense via the phagocyte NAPDH oxidase. Eros is required for expression of the NADPH oxidase components, gp91$\textit{phox}$ and p22$\textit{phox}$. Consequently, $\textit{Eros}$-deficient mice quickly succumb to infection. $\textit{Eros}$ also contributes to the formation of neutrophil extracellular traps (NETS) and impacts on the immune response to melanoma metastases. $\textit{Eros}$ is an ortholog of the plant protein Ycf4, which is necessary for expression of proteins of the photosynthetic photosystem 1 complex, itself also an NADPH oxio-reductase. We thus describe the key role of the previously uncharacterized protein Eros in host defense.D.C. Thomas was funded by a Wellcome Trust/CIMR Next Generation Fellowship, a National Institute for Health Research (NIHR) Clinical Lectureship, and a Starter Grant for Clinical Lecturers (Academy of Medical Sciences). K.G.C. Smith was funded by funded by the Medical Research Council (program grant MR/L019027) and is a Wellcome Investigator and a NIHR Senior Investigator. S. Clare and G. Dougan were funded by the Wellcome Trust (grant 098051). The Cambridge Institute for Medical Research is in receipt of a Wellcome Trust Strategic Award (079895). J.C.L is funded by a Wellcome Intermediate Clinical Fellowship 105920/2/14/2

Abstract B086: CRISPR-Cas9 engineering and screening in primary human T cells

Abstract Recent clinical data indicate immunotherapy can be an effective treatment for cancer patients, yielding dramatically increased survival times in some cases. As not all patients benefit from treatments such as anti-CTLA4 and anti-PD1 antibodies, a major focus in immuno-oncology research is finding new immuno-oncology targets, including those that alter the character and frequency of T-cell-mediated anti-tumor responses. We have had great success using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas9 mediated genome editing to probe gene function in cancer cells via the generation of knock-out and knock-in mutants. We are now applying this approach to primary immune cells and are deploying both CRISPR–Cas9 cell engineering and CRISPR–Cas9 screens to better understand T cell biology and to find new therapeutic targets. We use T cells negatively purified by magnetic sorting from peripheral blood mononuclear cells taken from healthy donors. Our initial work in primary human T cells has focused on the capacity to knock out and knock in genes using the Neon™ transfection system. Our data indicate that primary T cells are amenable to gene editing, and the capacity to rapidly modify loci, such as PDCD1, which encodes PD-1 enables generation of primary T cell models suitable for comprehending the function of modified receptor–ligand pairs involved in an immune checkpoint response. Our pooled sgRNA–Cas9 screens have used our in-house sgRNA libraries, which include a modified tracrRNA component improving Cas9 affinity and subsequently the performance of a typical sgRNA for promoting gene editing and modifying phenotype. Briefly, isolated CD3+ T cells are stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2. Next, the proliferating T cells are co-cultured with a GFP expressing Lentivirus that directs expression of Cas9 and an sgRNA drawn from a 3900 member sgRNA library targeting genes involved in the regulation of metabolism and a control library of 2442 guide RNAs. After prolonged Lentivirus and T cell co-culture, transduced T cells are sorted based on their GFP expression and periodically re-stimulated with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 to allow their proliferation and expansion over several weeks. Representative cell pellets are taken after GFP sorting and then at specific time points throughout the screen. For each time point, gDNA is extracted and PCR is carried out to isolate the gRNAs that are present in each cell and these are analysed using NGS. NGS results are interpreted using algorithms from the previously published model-based analysis of genome-wide CRISPR–Cas9 knockout (MAGeCK) and Bayesian normalisation of gene expression levels (BAGEL) approaches. These screens are currently ongoing and are being run in T cells isolated from five independent donors to assess the impact of donor variability. We will present results from these screens assessing guide drop-out kinetics and reproducibility by comparing the performance of specific guides over multiple time points in each of the donors. We anticipate that these data will be useful in building more complex screens that assess T cell biology in the presence of additional cells, such as myeloid derived suppressor cells, involved in regulating the immune response to tumor development and progression. Citation Format: Cristina Ghirelli, Thibault Laurent, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 engineering and screening in primary human T cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B086.</jats:p

Abstract 4123: CRISPR-Cas9 and siRNA screening in primary human immune cells

Abstract A major focus in immuno-oncology research is finding new immuno-oncology targets, including those that alter the character and frequency of T-cell-mediated anti-tumour responses. Screens using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9-mediated genome editing seem well placed to identify new targets. However, although CRISPR-Cas9 gene editing works well in primary T cells using electroporation, use of a lentivirus one vector system has proved challenging in primary T cells compared with cancer cell lines. We have used several different approaches to identify the most useful method for transduction of primary human T cells with CRISPR components. Electroporation of sgRNAs and mRNA encoding Cas9 into proliferating T cells efficiently generate T cells with specific gene knock-outs or knock-ins, with targeting rates of around 37% for gene knockout. Thus, primary T cells are amenable to CRISPR-Cas9 gene editing, and the capacity to rapidly modify loci enables generation of primary T cell models suitable for comprehending the function of modified receptor-ligand pairs involved in an immune checkpoint response. Our pooled sgRNA-Cas9 screens in cancer cell lines have used our in-house sgRNA libraries, which include a modified tracrRNA component improving Cas9 affinity and subsequently the performance of a typical sgRNA for promoting gene editing. However, use of the same approach in primary T cells has not resulted in efficient transduction of the library. Specifically, isolated CD3+ T cells stimulated in vitro with anti-CD3 and anti-CD28 antibodies in the presence of recombinant IL-2 resulted in no expression or low level expression of GFP after cells were transduced with a one vector CRISPR-Cas9 sgRNA library. Our experiments indicate, in line with published data, that T cells can be transduced effectively with lentivirus, thus we are examining the use of a two vector CRISPR-Cas9 system and the use of CRISPRi to idealise CRISPR screening in primary T cells. We are also carrying out target identification and validation in myeloid derived suppressor cells (MDSCs). We are using an siRNA approach in these cells, which are generated by PBMC co-culture with cancer cell lines for 7 days, or by culture in the presence of recombinant GM-CSF and IL-6 for 7 days. Our initial data indicate that these MDSCs can effectively suppress autologous, as well as allogeneic, CD8 T cell proliferation mediated by anti-CD3 and anti-CD28 stimulation and that siRNA knockdown is effective in MDSCs. We will use our druggable genome plus arrayed siRNA library to identify targets that when knocked down inhibit the capacity of MDSCs to suppress T cell proliferation. We anticipate that these data will be useful in identifying new targets that are involved in regulating an immune response to tumour development and progression. Citation Format: Cristina Ghirelli, Thibault Laurent, Simon Scrace, Kim Hoenderdos, Chris Lowe, Nicola McCarthy, Jonathan Moore. CRISPR-Cas9 and siRNA screening in primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4123. doi:10.1158/1538-7445.AM2017-4123</jats:p