Lack of PD-L1 Expression by iNKT Cells Improves the Course of Influenza A Infection

There is evidence indicating that invariant Natural Killer T (iNKT) cells play an important role in defense against influenza A virus (IAV). However, the effect of inhibitory receptor, programmed death-1 (PD-1), and its ligands, programmed death ligand (PD-L) 1 and 2 on iNKT cells in protection against IAV remains to be elucidated. Here we investigated the effects of these co-stimulatory molecules on iNKT cells in the response to influenza. We discovered that compare to the wild type, PD-L1 deficient mice show reduced sensitivity to IAV infection as evident by reduced weight loss, decreased pulmonary inflammation and cellular infiltration. In contrast, PD-L2 deficient mice showed augmented weight loss, pulmonary inflammation and cellular infiltration compare to the wild type mice after influenza infection. Adoptive transfer of iNKT cells from wild type, PD-L1 or PD-L2 deficient mice into iNKT cell deficient mice recapitulated these findings. Interestingly, in our transfer system PD-L1−/−-derived iNKT cells produced high levels of interferon-gamma whereas PD-L2−/−-derived iNKT cells produced high amounts of interleukin-4 and 13 suggesting a role for these cytokines in sensitivity to influenza. We identified that PD-L1 negatively regulates the frequency of iNKT cell subsets in the lungs of IAV infected mice. Altogether, these results demonstrate that lack of PD-L1 expression by iNKT cells reduces the sensitivity to IAV and that the presence of PD-L2 is important for dampening the deleterious inflammatory responses after IAV infection. Our findings potentially have clinical implications for developing new therapies for influenza

Gray platelet syndrome: proinflammatory megakaryocytes and α-granule loss cause myelofibrosis and confer metastasis resistance in mice.

NBEAL2 encodes a multidomain scaffolding protein with a putative role in granule ontogeny in human platelets. Mutations in NBEAL2 underlie gray platelet syndrome (GPS), a rare inherited bleeding disorder characterized by a lack of α-granules within blood platelets and progressive bone marrow fibrosis. We present here a novel Nbeal2(-/-) murine model of GPS and demonstrate that the lack of α-granules is due to their loss from platelets/mature megakaryocytes (MKs), and not by initial impaired formation. We show that the lack of Nbeal2 confers a proinflammatory phenotype to the bone marrow MKs, which in combination with the loss of proteins from α-granules drives the development of bone marrow fibrosis. In addition, we demonstrate that α-granule deficiency impairs platelet function beyond their purely hemostatic role and that Nbeal2 deficiency has a protective effect against cancer metastasis.This work was funded by the British Heart Foundation to CG (FS09/039) and WHO and AR (RG/09/12/28096); NHSBT to CB and HM; Wellcome Trust (WT098051) to ZM, ELC, JE, HWJ and AOS.This is the accepted manuscript. The final published version is available from Blood at http://www.bloodjournal.org/content/early/2014/09/25/blood-2014-04-566760

Synthetic lethality between PAXX and XLF in mammalian development.

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.Research in S.P.J.’s laboratory is funded by Cancer Research UK (CRUK) program grant number C6/A11224, the European Research Council, and the European Community Seventh Framework Programme grant agreement number HEALTH-F2-2010-259893 (DDResponse). Core funding is provided by CRUK (C6946/A14492) and the Wellcome Trust (WT092096). S.P.J. receives his salary from the University of Cambridge, UK, supplemented by CRUK. L.D.’s laboratory is funded by the Institut Pasteur as well as the European Research Council (ERC) under starting grant agreement number 310917. D.J.A.’s laboratory is supported by CRUK and the Wellcome Trust. A.N.B. is supported by a CRUK Career Development Fellowship (C29215/A20772).This is the final version of the article. It first appeared from Cold Spring Harbor Laboratory Press via https://doi.org/10.1101/gad.290510.11

CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation

Abstract Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.info:eu-repo/semantics/publishe

Interleukin-22 promotes phagolysosomal fusion to induce protection against Salmonella enterica Typhimurium in human epithelial cells.

Intestinal epithelial cells (IECs) play a key role in regulating immune responses and controlling infection. However, the direct role of IECs in restricting pathogens remains incompletely understood. Here, we provide evidence that IL-22 primed intestinal organoids derived from healthy human induced pluripotent stem cells (hIPSCs) to restrict Salmonella enterica serovar Typhimurium SL1344 infection. A combination of transcriptomics, bacterial invasion assays, and imaging suggests that IL-22-induced antimicrobial activity is driven by increased phagolysosomal fusion in IL-22-pretreated cells. The antimicrobial phenotype was absent in hIPSCs derived from a patient harboring a homozygous mutation in the IL10RB gene that inactivates the IL-22 receptor but was restored by genetically complementing the IL10RB deficiency. This study highlights a mechanism through which the IL-22 pathway facilitates the human intestinal epithelium to control microbial infection

FBXO7 sensitivity of phenotypic traits elucidated by a hypomorphic allele.

FBXO7 encodes an F box containing protein that interacts with multiple partners to facilitate numerous cellular processes and has a canonical role as part of an SCF E3 ubiquitin ligase complex. Mutation of FBXO7 is responsible for an early onset Parkinsonian pyramidal syndrome and genome-wide association studies have linked variants in FBXO7 to erythroid traits. A putative orthologue in Drosophila, nutcracker, has been shown to regulate the proteasome, and deficiency of nutcracker results in male infertility. Therefore, we reasoned that modulating Fbxo7 levels in a murine model could provide insights into the role of this protein in mammals. We used a targeted gene trap model which retained 4-16% residual gene expression and assessed the sensitivity of phenotypic traits to gene dosage. Fbxo7 hypomorphs showed regenerative anaemia associated with a shorter erythrocyte half-life, and male mice were infertile. Alterations to T cell phenotypes were also observed, which intriguingly were both T cell intrinsic and extrinsic. Hypomorphic mice were also sensitive to infection with Salmonella, succumbing to a normally sublethal challenge. Despite these phenotypes, Fbxo7 hypomorphs were produced at a normal Mendelian ratio with a normal lifespan and no evidence of neurological symptoms. These data suggest that erythrocyte survival, T cell development and spermatogenesis are particularly sensitive to Fbxo7 gene dosage

Alkaline ceramidase 1 is essential for mammalian skin homeostasis and regulating whole-body energy expenditure.

The epidermis is the outermost layer of skin that acts as a barrier to protect the body from the external environment and to control water and heat loss. This barrier function is established through the multistage differentiation of keratinocytes and the presence of bioactive sphingolipids such as ceramides, the levels of which are tightly regulated by a balance of ceramide synthase and ceramidase activities. Here we reveal the essential role of alkaline ceramidase 1 (Acer1) in the skin. Acer1-deficient (Acer1(-/-) ) mice showed elevated levels of ceramide in the skin, aberrant hair shaft cuticle formation and cyclic alopecia. We demonstrate that Acer1 is specifically expressed in differentiated interfollicular epidermis, infundibulum and sebaceous glands and consequently Acer1(-/-) mice have significant alterations in infundibulum and sebaceous gland architecture. Acer1(-/-) skin also shows perturbed hair follicle stem cell compartments. These alterations result in Acer1(-/-) mice showing increased transepidermal water loss and a hypermetabolism phenotype with associated reduction of fat content with age. We conclude that Acer1 is indispensable for mammalian skin homeostasis and whole-body energy homeostasis. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland

Combinatorial CRISPR screen identifies fitness effects of gene paralogues.

Genetic redundancy has evolved as a way for human cells to survive the loss of genes that are single copy and essential in other organisms, but also allows tumours to survive despite having highly rearranged genomes. In this study we CRISPR screen 1191 gene pairs, including paralogues and known and predicted synthetic lethal interactions to identify 105 gene combinations whose co-disruption results in a loss of cellular fitness. 27 pairs influence fitness across multiple cell lines including the paralogues FAM50A/FAM50B, two genes of unknown function. Silencing of FAM50B occurs across a range of tumour types and in this context disruption of FAM50A reduces cellular fitness whilst promoting micronucleus formation and extensive perturbation of transcriptional programmes. Our studies reveal the fitness effects of FAM50A/FAM50B in cancer cells