Sphingolipid imbalance is the culprit in a variety of neurological diseases, some affecting the myelin sheath. We have used whole exome sequencing in patients with undetermined leukoencephalopathies to uncover the endoplasmic reticulum lipid desaturase DEGS1 as the causative gene in nineteen patients from thirteen unrelated families. Shared features among the cases include severe motor arrest, early nystagmus, dystonia, spasticity and profound failure to thrive. MRI showed hypomyelination, thinning of corpus callosum and progressive thalami and cerebellar atrophy, suggesting a critical role of DEGS1 in myelin development and maintenance. This enzyme converts dihydroceramide (DhCer) into ceramide (Cer) in the final step of the de novo biosynthesis pathway. We detected a marked increase of the substrate DhCer and DhCer/Cer ratios in patient’s fibroblasts and muscle. Further, we used a knockdown approach for disease modelling in Danio rerio, followed by a preclinical test with the first-line treatment for multiple sclerosis, fingolimod (FTY720, Gilenya). The enzymatic inhibition of ceramide synthase, one step prior to DEGS1 in the pathway, by fingolimod, reduced the critical DhCer/Cer imbalance and the severe locomotor disability, increasing the number of myelinating oligodendrocytes in the zebrafish model. These proof-of-concept results pave the way to clinical translation.
Devesh C. Pant, Imen Dorboz, Agatha Schlüter, Stéphane Fourcade, Nathalie Launay, Javier Joya, Sergio Aguilera-Albesa, Maria Eugenia Yoldi, Carlos Casasnovas, Mary J. Willis, Montserrat Ruiz, Dorothée Ville, Gaetan Lesca, Karine Siquier-Pernet, Isabelle Desguerre, Huifang Yan, Jinming Wang, Margit Burmeister, Lauren Brady, Mark Tarnopolsky, Carles Cornet, Davide Rubbini, Javier Terriente, Kiely N. James, Damir Musaev, Maha S. Zaki, Marc C. Patterson, Brendan C. Lanpher, Eric W. Klee, Filippo Pinto e Vairo, Elizabeth Wohler, Nara Lygia de M. Sobreira, Julie S. Cohen, Reza Maroofian, Hamid Galehdari, Neda Mazaheri, Gholamreza Shariati, Laurence Colleaux, Diana Rodriguez, Joseph G. Gleeson, Cristina Pujades, Ali Fatemi, Odile Boespflug-Tanguy, Aurora Pujol
The development and function of stem and progenitor cells that produce blood cells are vital in physiology. GATA2 mutations cause GATA-2-deficiency syndrome involving immunodeficiency, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). GATA-2 physiological activities necessitate that it be strictly regulated, and cell type-specific enhancers fulfill this role. The +9.5 intronic enhancer harbors multiple conserved cis-elements, and germline mutations of these cis-elements are pathogenic in humans. Since mechanisms underlying how GATA2 enhancer disease mutations impact hematopoiesis and pathology are unclear, we generated mouse models of the enhancer mutations. While a multi-motif mutant was embryonic lethal, a single-nucleotide Ets motif mutant was viable, and steady-state hematopoiesis was normal. However, the Ets motif mutation abrogated stem/progenitor cell regeneration following stress. These results reveal a new mechanism in human genetics in which a disease predisposition mutation inactivates enhancer regenerative activity, while sparing developmental activity. Mutational sensitization to stress that instigates hematopoietic failure constitutes a paradigm for GATA-2-deficiency syndrome and other contexts of GATA-2-dependent pathogenesis.
Alexandra A. Soukup, Ye Zheng, Charu Mehta, Jun Wu, Peng Liu, Miao Cao, Inga Hofmann, Yun Zhou, Jing Zhang, Kirby D. Johnson, Kyunghee Choi, Sunduz Keles, Emery H. Bresnick
Genetic variants at the PTPN2 locus, which encodes the tyrosine phosphatase PTPN2, cause reduced gene expression and are linked to rheumatoid arthritis (RA) and other autoimmune diseases. PTPN2 inhibits signaling through the T cell and cytokine receptors and loss of PTPN2 promotes T cell expansion and CD4 and CD8-driven autoimmunity. However, it remains unknown whether loss of PTPN2 in FoxP3+ regulatory T cells (Treg) plays a role in autoimmunity. Here we aimed to model human autoimmune-predisposing PTPN2 variants, which results in a partial loss of PTPN2 expression, in mouse models of RA. We identified that reduced expression of Ptpn2 enhanced the severity of autoimmune arthritis in the T cell dependent SKG mouse model and demonstrated that this phenotype was mediated through a Treg-intrinsic mechanism. Mechanistically, we found that through dephosphorylation of STAT3, Ptpn2 inhibits IL-6-driven pathogenic loss of FoxP3 after Tregs have acquired RORγt expression, at a stage when chromatin accessibility for STAT3-targeted IL-17 associated transcription factors is maximized. We conclude that PTPN2 promotes FoxP3 stability in mouse RORγt+ Tregs and that loss of function of PTPN2 in Tregs contributes to the association between PTPN2 and autoimmunity.
Mattias N.D. Svensson, Karen M. Doody, Benjamin J. Schmiedel, Sourya Bhattacharyya, Bharat Panwar, Florian Wiede, Shen Yang, Eugenio Santelli, Dennis J. Wu, Cristiano Sacchetti, Ravindra Gujar, Grégory Seumois, William B. Kiosses, Isabelle Aubry, Gisen Kim, Piotr Mydel, Shimon Sakaguchi, Mitchell Kronenberg, Tony Tiganis, Michel L. Tremblay, Ferhat Ay, Pandurangan Vijayanand, Nunzio Bottini
Non-coding RNAs are emerging as important players in gene regulation and disease pathogeneses. Here, we show that a previously uncharacterized long non-coding RNA, NEXN-AS1, modulates the expression of the actin-binding protein NEXN and that NEXN exerts a protective role against atherosclerosis. An expression microarray analysis showed that the expression of both NEXN-AS1 and NEXN were reduced in human atherosclerotic plaques. In vitro experiments revealed that NEXN-AS1 interacted with the chromatin remodeler BAZ1A and the 5′-flanking region of the NEXN gene, and upregulated NEXN expression. Augmentation of NEXN-AS1 expression inhibited toll-like receptor-4 oligomerization and NFκB activity, downregulated the expression of adhesion molecules and inflammatory cytokines by endothelial cells, and suppressed monocyte adhesion to endothelial cells. These inhibitory effects of NEXN-AS1 were abolished by knockdown of NEXN. In vivo experiments of ApoE knockout mice fed a Western high-fat diet demonstrated that NEXN deficiency promoted atherosclerosis and increased macrophage abundance in atherosclerotic lesions, with heightened expression of adhesion molecules and inflammatory cytokines, whereas augmented NEXN expression deterred atherosclerosis. A group of patients with coronary artery disease were found to have lower blood NEXN levels than healthy individuals. These results indicate that NEXN-AS1 and NEXN represent potential therapeutic targets in atherosclerosis related diseases.
Yan-Wei Hu, Feng-Xia Guo, Yuan-Jun Xu, Pan Li, Zhi-Feng Lu, David G. McVey, Lei Zheng, Qian Wang, John H. Ye, Chun-Min Kang, Shao-Guo Wu, Jing-Jing Zhao, Xin Ma, Zhen Yang, Fu-Chun Fang, Yu-Rong Qiu, Bang-Ming Xu, Lei Xiao, Qian Wu, Li-Mei Wu, Li Ding, Tom R. Webb, Nilesh J. Samani, Shu Ye
The cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS/STING) pathway detects cytosolic DNA to activate innate immune responses. Poly(ADP-ribose) polymerase inhibitors (PARPi) selectively target cancer cells with DNA repair deficiencies such as those caused by BRCA1 mutations or ERCC1 defects. Using isogenic cell lines and patient-derived samples, we showed that ERCC1-defective non-small cell lung cancer (NSCLC) cells exhibit an enhanced type I interferon transcriptomic signature, and that low ERCC1 expression correlates with increased lymphocytic infiltration. We demonstrated that clinical PARPi, including olaparib and rucaparib, have cell-autonomous immunomodulatory properties in ERCC1-defective NSCLC and BRCA1-defective triple-negative breast cancer (TNBC) cells. Mechanistically, PARPi generated cytoplasmic chromatin fragments with micronuclei characteristics; these were found to activate cGAS/STING, downstream type I interferon signaling and CCL5 secretion. Importantly, these effects were suppressed in PARP1-null TNBC cells, suggesting that this phenotype resulted from an on-target effect of PARPi on PARP1. PARPi also potentiated interferon-γ-induced PD-L1 expression in NSCLC cell lines and in fresh patient tumor cells; this effect was enhanced in ERCC1-deficient contexts. Our data provide the preclinical rationale for using PARPi as immunomodulatory agents in appropriately molecularly-selected populations.
Roman M. Chabanon, Gareth Muirhead, Dragomir B. Krastev, Julien Adam, Daphné Morel, Marlène Garrido, Andrew Lamb, Clémence Hénon, Nicolas Dorvault, Mathieu Rouanne, Rebecca Marlow, Ilirjana Bajrami, Marta Llorca Cardeñosa, Asha Konde, Benjamin Besse, Alan Ashworth, Stephen J. Pettitt, Syed Haider, Aurélien Marabelle, Andrew N.J. Tutt, Jean-Charles Soria, Christopher J. Lord, Sophie Postel-Vinay
Mast cells (MCs) are immune sentinels but whether they also function as antigen-presenting cells (APCs) remains elusive. Using mouse models of MC-deficiency, we report MC-dependent recruitment and activation of multiple T cell subsets to the skin and draining lymph nodes (LNs) during dengue virus (DENV) infection. Newly-recruited and locally-proliferating γδT cells were the first responding T cell subset to MC-driven inflammation and their production of IFN-γ was MC-dependent. MC-γδ T cell conjugates were observed consistently in infected peripheral tissues, suggesting a new role for MCs as non-conventional APCs for γδT cells. MC-dependent γδT cell activation and proliferation during DENV infection required TCR signaling and the non-conventional antigen presentation molecule EPCR on MCs. γδT cells, not previously implicated in DENV host defense, killed infected target dendritic cells and contributed to clearance of DENV in vivo. We believe immune synapse formation between MCs and γδT cells is a novel mechanism to induce specific and protective immunity at sites of viral infection.
Chinmay Kumar Mantri, Ashley L. St. John
Joint pain is the defining symptom of osteoarthritis (OA) but its origin and mechanisms remain unclear. Here, we investigated an unprecedented role of osteoclast-initiated subchondral bone remodeling in sensory innervation for OA pain. We show that osteoclasts secrete NETRIN1 to induce sensory nerve axonal growth in subchondral bone. Reduction of osteoclast formation by knockout of receptor activator of nuclear factor kappa-B ligand (Rankl) in osteocytes inhibited the growth of sensory nerves into subchondral bone, DRG neuron hyperexcitability, and behavioral measures of pain hypersensitivity in OA mice. Moreover, we demonstrated a possible role for NETRIN1 secreted by osteoclasts during aberrant subchondral bone remodeling in inducing sensory innervation and OA pain through its receptor DCC (deleted in colorectal cancer). Importantly, knockout of Netrin1 in tartrate-resistant acid phosphatase (TRAP) positive osteoclasts or knockdown of Dcc reduces OA pain behavior. In particular, inhibition of osteoclast activity by alendronate modifies aberrant subchondral bone remodeling and reduces innervation and pain behavior at the early stage of OA. These results suggest that intervention of the axonal guidance molecules (e.g. NETRIN1) derived from aberrant subchondral bone remodeling may have therapeutic potential for OA pain.
Shouan Zhu, Jianxi Zhu, Gehua Zhen, Yihe Hu, Senbo An, Yusheng Li, Qin Zheng, Zhiyong Chen, Ya Yang, Mei Wan, Richard Leroy Skolasky, Yong Cao, Tianding Wu, Bo Gao, Mi Yang, Manman Gao, Julia Kuliwaba, Shuangfei Ni, Lei Wang, Chuanlong Wu, David Findlay, Holger K. Eltzschig, Hong Wei Ouyang, Janet Crane, Feng-Quan Zhou, Yun Guan, Xinzhong Dong, Xu Cao
Both natural influenza infection and current seasonal influenza vaccines primarily induce neutralising antibody responses against highly diverse epitopes within the “head” of the viral hemagglutinin (HA) protein. There is increasing interest on redirecting immunity towards the more conserved HA-stem or stalk as a means to broaden protective antibody responses. Here we examined HA-stem-specific B cell and T-follicular helper (Tfh) cell responses in the context of influenza infection and immunisation in mouse and monkey models. We found that during infection the stem domain was immunologically subdominant to the head in terms of serum antibody production and antigen-specific B and Tfh responses. Similarly, we found HA-stem immunogens were poorly immunogenic compared to the full-length HA with abolished sialic acid binding activity, with limiting Tfh elicitation a potential constraint to the induction or boosting of anti-stem immunity by vaccination. Finally, we confirm that currently licensed seasonal influenza vaccines can boost pre-existing memory responses against the HA-stem in humans. An increased understanding of the immune dynamics surrounding the HA-stem is essential to inform the design of next-generation influenza vaccines for broad and durable protection.
Hyon-Xhi Tan, Sinthujan Jegaskanda, Jennifer A. Juno, Robyn Esterbauer, Julius Wong, Hannah G. Kelly, Yi Liu, Danielle Tilmanis, Aeron C. Hurt, Jonathan W. Yewdell, Stephen J. Kent, Adam K. Wheatley
ARHGEF1 is a RhoA-specific guanine nucleotide exchange factor expressed in hematopoietic cells. We used whole-exome sequencing to identify compound heterozygous mutations in ARHGEF1, resulting in the loss of ARHGEF1 protein expression in two primary-antibody-deficient siblings presenting with recurrent severe respiratory tract infections and bronchiectasis. Both ARHGEF1-deficient patients showed an abnormal B cell immunophenotype, with a deficiency in marginal-zone and memory B cells and an increased frequency of transitional B cells. Furthermore, the patients’ blood contained immature myeloid cells. Analysis of a mediastinal lymph node from one patient highlighted the small size of the germinal centres and an abnormally high plasma cell content. On the molecular level, T and B lymphocytes from both patients displayed low RhoA activity and low steady-state actin polymerization (even after stimulation of lysophospholipid receptors). As a consequence of disturbed regulation of the RhoA downstream target ROCK, the patients’ lymphocytes failed to efficiently restrain AKT phosphorylation. Enforced ARHGEF1 expression or drug-induced activation of RhoA in patients’ cells corrected the impaired actin polymerization and AKT regulation. Our results indicate that ARHGEF1 activity in human lymphocytes is involved in controlling actin cytoskeleton dynamics, restraining PI3K/AKT signalling, and confining B lymphocytes and myelocytes within their dedicated functional environment.
Amine Bouafia, Sébastien Lofek, Julie Bruneau, Loïc Chentout, Hicham Lamrini, Amélie Trinquand, Marie-Céline Deau, Lucie Heurtier, Véronique Meignin, Capucine Picard, Elizabeth Macintyre, Olivier Alibeu, Marc Bras, Thierry Jo Molina, Marina Cavazzana, Isabelle André-Schmutz, Anne Durandy, Alain Fischer, Eric Oksenhendler, Sven Kracker
Energy stress, such as ischemia, induces mitochondrial damage and death in the heart. Degradation of damaged mitochondria by mitophagy is essential for the maintenance of healthy mitochondria and survival. Here we show that mitophagy during myocardial ischemia was mediated predominantly through autophagy characterized by Rab9-associated autophagosomes, rather than the well-characterized form of autophagy that is dependent upon the Atg-conjugation system and LC3. This form of mitophagy played an essential role in protecting the heart against ischemia and was mediated by a protein complex consisting of Ulk1, Rab9, Rip1 and Drp1. This complex allowed recruitment of trans-Golgi membranes associated with Rab9 to damaged mitochondria through Ser179 phosphorylation of Rab9 by Ulk1 and Ser616 phosphorylation of Drp1 by Rip1. Knock-in of Rab9 (S179A) abolished mitophagy and exacerbated injury in response to myocardial ischemia without affecting conventional autophagy. Mitophagy mediated through the Ulk1-Rab9-Rip1-Drp1 pathway protected the heart against ischemia by maintaining healthy mitochondria.
Toshiro Saito, Jihoon Nah, Shin-ichi Oka, Risa Mukai, Yoshiya Monden, Yusuhiro Maejima, Yoshiyuki Ikeda, Sebastiano Sciarretta, Tong Liu, Hong Li, Erdene Baljinnyam, Diego Fraidenraich, Luke Fritzky, Peiyong Zhai, Shizuko Ichinose, Mitsuaki Isobe, Chiao-Po Hsu, Mondira Kundu, Junichi Sadoshima
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