Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond–like phenotype.
Raphael Carapito, Martina Konantz, Catherine Paillard, Zhichao Miao, Angélique Pichot, Magalie S. Leduc, Yaping Yang, Katie L. Bergstrom, Donald H. Mahoney, Deborah L. Shardy, Ghada Alsaleh, Lydie Naegely, Aline Kolmer, Nicodème Paul, Antoine Hanauer, Véronique Rolli, Joëlle S. Müller, Elisa Alghisi, Loïc Sauteur, Cécile Macquin, Aurore Morlon, Consuelo Sebastia Sancho, Patrizia Amati-Bonneau, Vincent Procaccio, Anne-Laure Mosca-Boidron, Nathalie Marle, Naël Osmani, Olivier Lefebvre, Jacky G. Goetz, Sule Unal, Nurten A. Akarsu, Mirjana Radosavljevic, Marie-Pierre Chenard, Fanny Rialland, Audrey Grain, Marie-Christine Béné, Marion Eveillard, Marie Vincent, Julien Guy, Laurence Faivre, Christel Thauvin-Robinet, Julien Thevenon, Kasiani Myers, Mark D. Fleming, Akiko Shimamura, Elodie Bottollier-Lemallaz, Eric Westhof, Claudia Lengerke, Bertrand Isidor, Seiamak Bahram
NK cells, lymphocytes of the innate immune system, are important for defense against infectious pathogens and cancer. Classically, the CD56dim NK cell subset is thought to mediate antitumor responses, whereas the CD56bright subset is involved in immunomodulation. Here, we challenge this paradigm by demonstrating that brief priming with IL-15 markedly enhanced the antitumor response of CD56bright NK cells. Priming improved multiple CD56bright cell functions: degranulation, cytotoxicity, and cytokine production. Primed CD56bright cells from leukemia patients demonstrated enhanced responses to autologous blasts in vitro, and primed CD56bright cells controlled leukemia cells in vivo in a murine xenograft model. Primed CD56bright cells from multiple myeloma (MM) patients displayed superior responses to autologous myeloma targets, and furthermore, CD56bright NK cells from MM patients primed with the IL-15 receptor agonist ALT-803 in vivo displayed enhanced ex vivo functional responses to MM targets. Effector mechanisms contributing to IL-15–based priming included improved cytotoxic protein expression, target cell conjugation, and LFA-1–, CD2-, and NKG2D-dependent activation of NK cells. Finally, IL-15 robustly stimulated the PI3K/Akt/mTOR and MEK/ERK pathways in CD56bright compared with CD56dim NK cells, and blockade of these pathways attenuated antitumor responses. These findings identify CD56bright NK cells as potent antitumor effectors that warrant further investigation as a cancer immunotherapy.
Julia A. Wagner, Maximillian Rosario, Rizwan Romee, Melissa M. Berrien-Elliott, Stephanie E. Schneider, Jeffrey W. Leong, Ryan P. Sullivan, Brea A. Jewell, Michelle Becker-Hapak, Timothy Schappe, Sara Abdel-Latif, Aaron R. Ireland, Devika Jaishankar, Justin A. King, Ravi Vij, Dennis Clement, Jodie Goodridge, Karl-Johan Malmberg, Hing C. Wong, Todd A. Fehniger
Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its γ-carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function.
Omar El-Rifai, Jacqueline Chow, Julie Lacombe, Catherine Julien, Denis Faubert, Delia Susan-Resiga, Rachid Essalmani, John W.M. Creemers, Nabil G. Seidah, Mathieu Ferron
Atherosclerosis is the underlying etiology of cardiovascular disease, the leading cause of death worldwide. Atherosclerosis is a heterogeneous disease in which only a small fraction of lesions lead to heart attack, stroke, or sudden cardiac death. A distinct type of plaque containing large necrotic cores with thin fibrous caps often precipitates these acute events. Here, we show that Ca2+/calmodulin-dependent protein kinase γ (CaMKIIγ) in macrophages plays a major role in the development of necrotic, thin-capped plaques. Macrophages in necrotic and symptomatic atherosclerotic plaques in humans as well as advanced atherosclerotic lesions in mice demonstrated activation of CaMKII. Western diet–fed LDL receptor–deficient (Ldlr–/–) mice with myeloid-specific deletion of CaMKII had smaller necrotic cores with concomitantly thicker collagen caps. These lesions demonstrated evidence of enhanced efferocytosis, which was associated with increased expression of the macrophage efferocytosis receptor MerTK. Mechanistic studies revealed that CaMKIIγ-deficient macrophages and atherosclerotic lesions lacking myeloid CaMKIIγ had increased expression of the transcription factor ATF6. We determined that ATF6 induces liver X receptor-α (LXRα), an Mertk-inducing transcription factor, and that increased MerTK expression and efferocytosis in CaMKIIγ-deficient macrophages is dependent on LXRα. These findings identify a macrophage CaMKIIγ/ATF6/LXRα/MerTK pathway as a key factor in the development of necrotic atherosclerotic plaques.
Amanda C. Doran, Lale Ozcan, Bishuang Cai, Ze Zheng, Gabrielle Fredman, Christina C. Rymond, Bernhard Dorweiler, Judith C. Sluimer, Joanne Hsieh, George Kuriakose, Alan R. Tall, Ira Tabas
Netrin-1 is a secreted protein that was first identified 20 years ago as an axon guidance molecule that regulates midline crossing in the CNS. It plays critical roles in various tissues throughout development and is implicated in tumorigenesis and inflammation in adulthood. Despite extensive studies, no inherited human disease has been directly associated with mutations in NTN1, the gene coding for netrin-1. Here, we have identified 3 mutations in exon 7 of NTN1 in 2 unrelated families and 1 sporadic case with isolated congenital mirror movements (CMM), a disorder characterized by involuntary movements of one hand that mirror intentional movements of the opposite hand. Given the diverse roles of netrin-1, the absence of manifestations other than CMM in NTN1 mutation carriers was unexpected. Using multimodal approaches, we discovered that the anatomy of the corticospinal tract (CST) is abnormal in patients with NTN1-mutant CMM. When expressed in HEK293 or stable HeLa cells, the 3 mutated netrin-1 proteins were almost exclusively detected in the intracellular compartment, contrary to WT netrin-1, which is detected in both intracellular and extracellular compartments. Since netrin-1 is a diffusible extracellular cue, the pathophysiology likely involves its loss of function and subsequent disruption of axon guidance, resulting in abnormal decussation of the CST.
Aurélie Méneret, Elizabeth A. Franz, Oriane Trouillard, Thomas C. Oliver, Yvrick Zagar, Stephen P. Robertson, Quentin Welniarz, R.J. MacKinlay Gardner, Cécile Gallea, Myriam Srour, Christel Depienne, Christine L. Jasoni, Caroline Dubacq, Florence Riant, Jean-Charles Lamy, Marie-Pierre Morel, Raphael Guérois, Jessica Andreani, Coralie Fouquet, Mohamed Doulazmi, Marie Vidailhet, Guy A. Rouleau, Alexis Brice, Alain Chédotal, Isabelle Dusart, Emmanuel Roze, David Markie
In psoriasis, an IL-17–mediated inflammatory skin disease, skin lesions resolve with therapy, but often recur in the same locations when therapy is discontinued. We propose that residual T cell populations in resolved psoriatic lesions represent the pathogenic T cells of origin in this disease. Utilizing high-throughput screening (HTS) of the T cell receptor (TCR) and immunostaining, we found that clinically resolved psoriatic lesions contained oligoclonal populations of T cells that produced IL-17A in both resolved and active psoriatic lesions. Putative pathogenic clones preferentially utilized particular Vβ and Vα subfamilies. We identified 15 TCRβ and 4 TCRα antigen receptor sequences shared between psoriasis patients and not observed in healthy controls or other inflammatory skin conditions. To address the relative roles of αβ versus γδ T cells in psoriasis, we carried out TCR/δ HTS. These studies demonstrated that the majority of T cells in psoriasis and healthy skin are αβ T cells. γδ T cells made up 1% of T cells in active psoriasis, less than 1% in resolved psoriatic lesions, and less than 2% in healthy skin. All of the 70 most frequent putative pathogenic T cell clones were αβ T cells. In summary, IL-17–producing αβ T cell clones with psoriasis-specific antigen receptors exist in clinically resolved psoriatic skin lesions. These cells likely represent the disease-initiating pathogenic T cells in psoriasis, suggesting that lasting control of this disease will require suppression of these resident T cell populations.
Tiago R. Matos, John T. O’Malley, Elizabeth L. Lowry, David Hamm, Ilan R. Kirsch, Harlan S. Robins, Thomas S. Kupper, James G. Krueger, Rachael A. Clark
In multiple sclerosis, the pathological interaction between autoreactive Th cells and mononuclear phagocytes in the CNS drives initiation and maintenance of chronic neuroinflammation. Here, we found that intrathecal transplantation of neural stem/precursor cells (NPCs) in mice with experimental autoimmune encephalomyelitis (EAE) impairs the accumulation of inflammatory monocyte-derived cells (MCs) in the CNS, leading to improved clinical outcome. Secretion of IL-23, IL-1, and TNF-α, the cytokines required for terminal differentiation of Th cells, decreased in the CNS of NPC-treated mice, consequently inhibiting the induction of GM-CSF–producing pathogenic Th cells. In vivo and in vitro transcriptome analyses showed that NPC-secreted factors inhibit MC differentiation and activation, favoring the switch toward an antiinflammatory phenotype. Tgfb2–/– NPCs transplanted into EAE mice were ineffective in impairing MC accumulation within the CNS and failed to drive clinical improvement. Moreover, intrathecal delivery of TGF-β2 during the effector phase of EAE ameliorated disease severity. Taken together, these observations identify TGF-β2 as the crucial mediator of NPC immunomodulation. This study provides evidence that intrathecally transplanted NPCs interfere with the CNS-restricted inflammation of EAE by reprogramming infiltrating MCs into antiinflammatory myeloid cells via secretion of TGF-β2.
Donatella De Feo, Arianna Merlini, Elena Brambilla, Linda Ottoboni, Cecilia Laterza, Ramesh Menon, Sundararajan Srinivasan, Cinthia Farina, Jose Manuel Garcia Manteiga, Erica Butti, Marco Bacigaluppi, Giancarlo Comi, Melanie Greter, Gianvito Martino
Escherichia coli and other Enterobacteriaceae are among the most common pathogens of the human urinary tract. Among the genetic gains of function associated with urinary E. coli isolates is the Yersinia high pathogenicity island (HPI), which directs the biosynthesis of yersiniabactin (Ybt), a virulence-associated metallophore. Using a metabolomics approach, we found that E. coli and other Enterobacteriaceae expressing the Yersinia HPI also secrete escherichelin, a second metallophore whose chemical structure matches a known synthetic inhibitor of the virulence-associated pyochelin siderophore system in Pseudomonas aeruginosa. We detected escherichelin during clinical E. coli urinary tract infection (UTI) and experimental human colonization with a commensal, potentially probiotic E. coli bacteriuria strain. Escherichelin production by colonizing enterobacteria may help human hosts resist opportunistic infections by Pseudomonas and other pyochelin-expressing bacteria. This siderophore-based mechanism of microbial antagonism may be one of many elements contributing to the protective effects of the human microbiome. Future UTI-preventive probiotic strains may benefit by retaining the escherichelin biosynthetic capacity of the Yersinia HPI while eliminating the Ybt biosynthetic capacity.
Shannon I. Ohlemacher, Daryl E. Giblin, D. André d’Avignon, Ann E. Stapleton, Barbara W. Trautner, Jeffrey P. Henderson
Consumption of human breast milk (HBM) attenuates the incidence of necrotizing enterocolitis (NEC), which remains a leading and intractable cause of mortality in preterm infants. Here, we report that this diminution correlates with alterations in the gut microbiota, particularly enrichment of Propionibacterium species. Transfaunation of microbiota from HBM-fed preterm infants or a newly identified and cultured Propionibacterium strain, P. UF1, to germfree mice conferred protection against pathogen infection and correlated with profound increases in intestinal Th17 cells. The induction of Th17 cells was dependent on bacterial dihydrolipoamide acetyltransferase (DlaT), a major protein expressed on the P. UF1 surface layer (S-layer). Binding of P. UF1 to its cognate receptor, SIGNR1, on dendritic cells resulted in the regulation of intestinal phagocytes. Importantly, transfer of P. UF1 profoundly mitigated induced NEC-like injury in neonatal mice. Together, these results mechanistically elucidate the protective effects of HBM and P. UF1–induced immunoregulation, which safeguard against proinflammatory diseases, including NEC.
Natacha Colliou, Yong Ge, Bikash Sahay, Minghao Gong, Mojgan Zadeh, Jennifer L. Owen, Josef Neu, William G. Farmerie, Francis Alonzo III, Ken Liu, Dean P. Jones, Shuzhao Li, Mansour Mohamadzadeh
Despite its central position in oncogenic intracellular signaling networks, the role of mTORC1 in epithelial development has not been studied extensively in vivo. Here, we have used the epidermis as a model system to elucidate the cellular effects and signaling feedback sequelae of mTORC1 loss of function in epithelial tissue. In mice with conditional epidermal loss of the mTORC1 components Rheb or Rptor, mTORC1 loss of function unexpectedly resulted in a profound skin barrier defect with epidermal abrasions, blistering, and early postnatal lethality, due to a thinned epidermis with decreased desmosomal protein expression and incomplete biochemical differentiation. In mice with mTORC1 loss of function, we found that Rho kinase (ROCK) signaling was constitutively activated, resulting in increased cytoskeletal tension and impaired cell-cell adhesion. Inhibition or silencing of ROCK1 was sufficient to rescue keratinocyte adhesion and biochemical differentiation in these mice. mTORC1 loss of function also resulted in marked feedback upregulation of upstream TGF-β signaling, triggering ROCK activity and its downstream effects on desmosomal gene expression. These findings elucidate a role for mTORC1 in the regulation of epithelial barrier formation, cytoskeletal tension, and cell adhesion, underscoring the complexity of signaling feedback following mTORC1 inhibition.
Kaushal Asrani, Akshay Sood, Alba Torres, Dan Georgess, Pornima Phatak, Harsimar Kaur, Amber Dubin, C. Conover Talbot Jr., Loubna Elhelu, Andrew J. Ewald, Bo Xiao, Paul Worley, Tamara L. Lotan