Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin-inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated, conventional CD4+ T cells (Tconv) and proinflammatory dendritic cells (DC); which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83+ target cells, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show human, acute myeloid leukemia (AML) expresses CD83 and myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to prevent two critical complications of allo-HCT; GVHD and relapse. Thus, human CD83 CAR T cells warrant clinical investigation in GVHD prevention and treatment, as well as targeting CD83+ AML.
Bishwas Shrestha, Kelly Walton, Jordan Reff, Elizabeth M. Sagatys, Nhan Tu, Justin C. Boucher, Gongbo Li, Tayyeb Ghafoor, Martin Felices, Jeffrey Miller, Joseph Pidala, Bruce R. Blazar, Claudio Anasetti, Brian C. Betts, Marco L. Davila
Despite the widespread use of antibiotics, bacterial pneumonias in donors strongly predispose to the fatal syndrome of primary graft dysfunction (PGD) following lung transplantation. We report that bacterial endotoxin persists in human donor lungs after pathogen is cleared with antibiotics and is associated with neutrophil infiltration and PGD. In mouse models, depletion of tissue-resident alveolar macrophages (TRAM) attenuated neutrophil recruitment in response to endotoxin as shown by compartmental staining and intravital imaging. Bone marrow chimeric mice revealed that neutrophils were recruited by TRAM through activation of TLR4 in a MyD88-dependent manner. Intriguingly, low levels of endotoxin, insufficient to cause donor lung injury, promoted TRAM-dependent production of CXCL2, increased neutrophil recruitment, and led to PGD, which was independent of donor non-classical monocytes. Reactive oxygen species (ROS) increased in human donor lungs starting from the warm-ischemia phase and were associated with increased transcription and translocation to the plasma membrane of TLR4 in donor TRAM. Consistently, scavenging ROS or inhibiting their production to prevent TLR4 transcription/translocation or blockade of TLR4 or co-receptor CD14 on donor TRAM prevented neutrophil recruitment in response to endotoxin and ameliorated PGD. Our studies demonstrate that residual endotoxin after successful treatment of donor bacterial pneumonia promotes PGD through ischemia-reperfusion-primed donor TRAM..
Mahzad Akbarpour, Emilia Lecuona, Stephen Chiu, Qiang Wu, Melissa Querrey, Ramiro Fernandez, Felix Luis Nunez-Santana, Haiying Sun, Sowmya Ravi, Chitaru Kurihara, James M. Walter, Nikita Joshi, Ziyou Ren, Scott C. Roberts, Alan R. Hauser, Daniel Kreisel, Wenjun Li, Navdeep Chandel, Alexander V. Misharin, Thalachallour Mohanakumar, G.R. Scott Budinger, Ankit Bharat
Background: Bariatric surgeries are the most effective treatments for successful and sustained weight loss but individuals vary in treatment response. Understanding the neurobiological and behavioral mechanisms accounting for this variation could lead to the development of personalized therapeutic approaches and improve treatment outcomes. The primary objectives were to investigate changes in taste preferences and taste-induced brain responses after Roux-en-Y gastric bypass (RYGB) and vertical sleeve gastrectomy (VSG) and to identify potential taste-related predictors of weight loss. Methods: Women, ages 18 to 55, with a body mass index ≥ 35 kg/m2 and approved for bariatric surgery at the Johns Hopkins Center for Bariatric Surgery were recruited for participation. Demographics, anthropometrics, liking ratings, and neural responses to varying concentrations of sucrose+fat mixtures were assessed pre- and post-surgery via visual analogue scales and functional magnetic resonance imaging. Results: Bariatric surgery produced decreases in liking for sucrose-sweetened mixtures. Greater preference for sucrose-sweetened mixtures prior to surgery was associated with greater weight loss in RYGB but not VSG. In the RYGB group only, individuals who showed lower taste-induced activation in the ventral tegmental area (VTA) prior to surgery and greater changes in taste-induced VTA activation two weeks following surgery experienced better weight loss. Conclusions: The anatomical and/or metabolic changes associated with RYGB may more effectively “reset” the neural processing of reward stimuli, thereby rescuing the blunted activation in the mesolimbic pathway found in patients with obesity. Further, these findings suggest that RYGB may be particularly effective in patients with a preference for sweet foods. Trial Registration: Not Applicable.Funding: K23DK100559 and The Dalio Philanthropies. Funding: K23DK100559 and The Dalio Philanthropies.
Kimberly R. Smith, Afroditi Papantoni, Maria G. Veldhuizen, Vidyulata Kamath, Civonnia Harris, Timothy H. Moran, Susan Carnell, Kimberley E. Steele
Understanding tumor resistance to T cell immunotherapies is critical to improve patient outcomes. Our study revealed a role for transcriptional suppression of the tumor-intrinsic HLA class I (HLA-I) antigen processing and presentation machinery (APM) in therapy resistance. Low HLA-I APM mRNA levels in melanoma metastases prior to immune checkpoint blockade (ICB) correlated with non-responsiveness to therapy and poor clinical outcome. Patient-derived melanoma cells with silenced HLA-I APM escaped recognition by autologous CD8+ T cells. However, targeted activation of the innate immunoreceptor RIG-I initiated de novo HLA-I APM transcription thereby overcoming T cell resistance. Antigen presentation was restored in interferon (IFN)-sensitive but also immunoedited IFN-resistant melanoma models through RIG-I-dependent stimulation of an IFN-independent salvage pathway involving IRF1 and IRF3. Likewise, enhanced HLA-I APM expression was detected in RIG-I (DDX58)-high melanoma biopsies, correlating with improved patient survival. Induction of HLA-I APM by RIG-I synergized with antibodies blocking PD-1 and TIGIT inhibitory checkpoints in boosting the anti-tumor T cell activity of ICB non-responders. Overall, the herein identified IFN-independent effect of RIG-I on tumor antigen presentation and T cell recognition proposes innate immunoreceptor targeting as a strategy to overcome intrinsic T cell resistance of IFN-sensitive and IFN-resistant melanomas and improve clinical outcomes in immunotherapy.
Lina Such, Fang Zhao, Derek Liu, Beatrice Thier, Vu Thuy Khanh Le-Trilling, Antje Sucker, Christoph Coch, Natalia Pieper, Sebastian Howe, Hilal Bhat, Halime Kalkavan, Cathrin Ritter, Robin Brinkhaus, Selma Ugurel, Johannes Köster, Ulrike Seifert, Ulf Dittmer, Martin Schuler, Karl Sebastian Lang, Thomas A Kufer, Gunther Hartmann, Jürgen Christian Becker, Susanne Horn, Soldano Ferrone, David Liu, Eliezer M. Van Allen, Dirk Schadendorf, Klaus Griewank, Mirko Trilling, Annette Paschen
Dominant mutations in the HSP70 co-chaperone DNAJB6 cause a late onset muscle disease termed limb girdle muscular dystrophy type D1 (LGMDD1), which is characterized by protein aggregation and vacuolar myopathology. Disease mutations reside within the G/F domain of DNAJB6, but the molecular mechanisms underlying dysfunction are not well understood. Using yeast, cell culture, and mouse models of LGMDD1, we found that the toxicity associated with disease-associated DNAJB6 required its interaction with HSP70, and that abrogating this interaction genetically or with small molecules was protective. In skeletal muscle, DNAJB6 localizes to the Z-disc with HSP70. Whereas HSP70 normally diffused rapidly between the Z-disc and sarcoplasm, the rate of HSP70’s diffusion in LGMDD1 mouse muscle was diminished likely because it has an unusual affinity for the Z-disc and mutant DNAJB6. Treating LGMDD1 mice with a small molecule inhibitor of the DNAJ-HSP70 complex re-mobilized HSP70, improved strength and corrected myopathology. These data support a model in which LGMDD1 mutations in DNAJB6 are a gain-of-function disease that is, counter-intuitively, mediated via HSP70 binding. Thus, therapeutic approaches targeting HSP70:DNAJB6 may be effective in treating this inherited muscular dystrophy.
Rocio Bengoechea, Andrew R. Findlay, Ankan K. Bhadra, Hao Shao, Kevin C. Stein, Sara K. Pittman, Jill Daw, Jason E. Gestwicki, Heather L. True, Conrad C. Weihl
While Canakinumab Anti-Inflammatory Thrombosis Outcomes Study (CANTOS) established the role of treating inflammation in atherosclerosis, our understanding of endothelial activation at atherosclerosis-prone sites remains limited. Disturbed flow at atheroprone regions primes plaque inflammation by enhancing endothelial NF-κB signaling. Herein, we demonstrate a role for the Nck adaptor proteins in disturbed flow-induced endothelial activation. Although highly similar, only Nck1 deletion, but not Nck2 deletion, limited flow-induced NF-κB activation and proinflammatory gene expression. Nck1 knockout mice showed reduced endothelial activation and inflammation in both models of disturbed flow and high fat diet-induced atherosclerosis, whereas Nck2 deletion did not. Bone marrow chimeras confirmed that vascular Nck1, but not hematopoietic Nck1, mediated this effect. Domain swap experiments and point mutations identified the Nck1 SH2 domain and the first SH3 domain as critical for flow-induced endothelial activation. We further characterized Nck1’s proinflammatory role by identifying interleukin-1 type I receptor kinase-1 (IRAK-1) as a Nck1-selective binding partner, demonstrating IRAK-1 activation by disturbed flow required Nck1 in vitro and in vivo, showing endothelial Nck1 and IRAK-1 staining in early human atherosclerosis, and demonstrating that disturbed flow-induced endothelial activation required IRAK-1. Taken together, our data reveal a hitherto unknown link between Nck1 and IRAK-1 in atherogenic inflammation.
Mabruka Alfaidi, Christina H. Acosta, Dongdong Wang, James G. Traylor, A. Wayne Orr
Esophageal atresia (EA/TEF) are common congenital abnormalities of the gastrointestinal tract. The etiology of EA/TEF is not well understood. We hypothesized that EA/TEF may be the direct consequence of abnormal expression of Noggin (NOG) signaling cascade. Here we showed that, in neonates with EA/TEF, NOG was missing from the atretic esophagus, resulting in immature esophagus that contains respiratory glands, and cilia. When using mouse esophageal organoid units (EOUs) or tracheal organoid units (TOU) as a model of foregut development in vitro, NOG determined the fate of foregut progenitors by allowing expression of esophageal epithelium proteins. When NOG was present in the culture of mTOU, it altered the cell morphology of the organoid unit epithelium, allowing expression of squamous cell proteins normally found in esophagus. On the other hand, when NOG was inhibited in mEOU, the organoid epithelium began to express respiratory markers mimicking the phenotype seen in pathology samples of human EA/TEF. Moreover, human EOU derived from EA/TEF patients were small, fibrotic and lack esophageal epithelium, but when NOG was added, the EOU grew larger, healthier and express esophageal proteins. These results indicate that Noggin is a critical regulator of cell fate decisions between esophageal and pulmonary morphogenesis.
Carolina Pinzon-Guzman, Sreedhara Sangadala, Katherine M. Riera, Evgenya Y. Popova, Elizabeth Manning, Won Jae Huh, Matthew S. Alexander, Julia S. Shelton, Scott D. Boden, James R. Goldenring
Aberrant, neovascular retinal blood vessel growth is a vision-threatening complication in ischemic retinal diseases. It is driven by retinal hypoxia frequently caused by capillary non-perfusion and endothelial cell (EC) loss. We investigated the role of EC apoptosis in this process using a mouse model of ischemic retinopathy, in which vessel closure and EC apoptosis cause capillary regression and retinal ischemia followed by neovascularisation. Protecting ECs from apoptosis in this model did not prevent capillary closure or retinal ischemia. Nonetheless, it prevented the clearance of ECs from closed capillaries, delaying vessel regression and allowing ECs to persist in clusters throughout the ischemic zone. In response to hypoxia, these preserved ECs underwent a vessel sprouting response and rapidly reassembled into a functional vascular network. This alleviated retinal hypoxia, preventing subsequent pathogenic neovascularisation. Vessel reassembly was not limited by VEGFA neutralisation, suggesting it was not dependent on the excess VEGFA produced by the ischemic retina. Neutralisation of ANG2 did not prevent vessel reassembly, but did impair subsequent angiogenic expansion of the reassembled vessels. Blockade of EC apoptosis may promote ischemic tissue re-vascularisation by preserving ECs within ischemic tissue that retain the capacity to reassemble a functional network and rapidly restore blood supply.
Zoe L. Grant, Lachlan Whitehead, Vickie H. Y. Wong, Zheng He, Richard Y. Yan, Abigail R. Miles, Andrew V. Benest, David O. Bates, Claudia Prahst, Katie Bentley, Bang V. Bui, Robert C.A. Symons, Leigh Coultas
Breast cancer stem cells (BCSCs) play a critical role in cancer recurrence and metastasis. Chemotherapy induces BCSC specification through increased expression of pluripotency factors, but how their expression is regulated is not fully understood. Here, we delineate a hypoxia-inducible factor 1 (HIF-1)-controlled pathway that epigenetically activates pluripotency factor gene transcription in response to chemotherapy. Paclitaxel induces HIF-1-dependent expression of S100A10, which forms a complex with ANXA2 that interacts with histone chaperone SPT6 and histone demethylase KDM6A. S100A10, ANXA2, SPT6, and KDM6A are recruited to OCT4 binding sites and KDM6A erases H3K27me3 chromatin marks, facilitating transcription of genes encoding the pluripotency factors NANOG, SOX2, and KLF4, which along with OCT4 are responsible for BCSC specification. Silencing of S100A10, ANXA2, SPT6, or KDM6A expression blocks chemotherapy-induced enrichment of BCSCs, impairs tumor initiation, and increases time to tumor recurrence after chemotherapy is discontinued. Pharmacological inhibition of KDM6A also impairs chemotherapy-induced BCSC enrichment. These results suggest that targeting HIF-1/S100A10-dependent and KDM6A-mediated epigenetic activation of pluripotency factor gene expression in combination with chemotherapy may block BCSC enrichment and improve clinical outcome.
Haiquan Lu, Yangyiran Xie, Linh Tran, Jie Lan, Yongkang Yang, Naveena L. Murugan, Ru Wang, Yueyang J. Wang, Gregg L. Semenza
Despite recent advances in understanding chronic inflammation remission, global analyses have not been explored to systematically discover genes or pathways underlying the resolution dynamics of chronic inflammatory diseases. Here, we performed time-course gene expression profiling of mouse synovial tissues along progression and resolution of collagen-induced arthritis (CIA) and identified genes associated with inflammation resolution. Through network analysis of these genes, we predicted three key secretory factors responsible for the resolution of CIA: Itgb1, Rps3, and Ywhaz. These factors were predominantly expressed by regulatory T cells and anti-inflammatory M2 macrophages, suppressing production of pro-inflammatory cytokines. In particular, Ywhaz was elevated in the sera of mice with arthritis resolution and in the urine of rheumatoid arthritis (RA) patients with good therapeutic responses. Moreover, adenovirus-mediated transfer of the Ywhaz gene to the affected joints substantially inhibited arthritis progression in mice with CIA and suppressed expression of pro-inflammatory cytokines in joint tissues, lymph nodes, and spleens, suggesting Ywhaz as an excellent target for RA therapy. Therefore, our comprehensive analysis of dynamic synovial transcriptomes provides previously unidentified anti-arthritic genes, Itgb1, Rps3, and Ywhaz, which can serve as molecular markers to predict disease remission, as well as therapeutic targets for chronic inflammatory arthritis.
Jin-Sun Kong, Ji-Hwan Park, Seung-Ah Yoo, Ki-Myo Kim, Yeung-Jin Bae, Yune-Jung Park, Chul-Soo Cho, Daehee Hwang, Wan-Uk Kim
Mothers living near high-traffic roads before or during pregnancy have increased odds of having children with asthma. Mechanisms are unknown. Using a mouse model, here we showed that maternal exposure to diesel exhaust particles (DEP) predisposed offspring to allergic airway disease/AAD (murine counterpart of human asthma) through programming of their NK cells; predisposition to AAD did not develop in ‘DEP’ pups that lacked NK cells and was induced in normal pups receiving NK cells from wild type ‘DEP’ pups. “DEP’ NK cells expressed GATA3 and co-secreted IL-13 and the ‘killer’ protease granzyme B in response to allergen challenge. Extracellular granzyme B did not kill but instead it stimulated protease-activated receptor 2 (PAR2) to cooperate with IL-13 in the induction of IL-25 in airway epithelial cells. Through loss-of-function and reconstitution experiments in pups, we showed that NK cells and granzyme B were required for IL-25 induction and activation of the type-2 immune response, and IL-25 mediated NK cell effects on type-2 response and AAD. Lastly, experiments using human cord blood and airway epithelial cells suggested that DEP might induce an identical pathway in humans. Collectively, we described an NK cell-dependent endotype of AAD that emerged in early life as a result of maternal exposure to DEP.
Qian Qian, Bidisha Paul Chowdhury, Zehua Sun, Jerica Lenberg, Rafeul Alam, Eric Vivier, Magdalena M. Gorska
The identification of loss-of-function mutations in MKRN3 in patients with central precocious puberty (CPP) in association with the decrease in MKRN3 expression in the medial basal hypothalamus (MBH) of mice prior to the initiation of reproductive maturation suggest that MKRN3 is acting as a ‘brake’ on GnRH secretion during childhood. In the current study, we investigated the mechanism by which MKRN3 prevents premature manifestation of the pubertal process. We showed that, as in mice, MKRN3 expression is high in the hypothalamus of rats and nonhuman primates early in life, declining as puberty approaches, and is independent of sex steroid hormones. We demonstrated that Mkrn3 is expressed in Kiss1 neurons of the mouse hypothalamic arcuate nucleus (ARC) and that MKRN3 repressed promoter activity of human KISS1 and TAC3, two key stimulators of GnRH secretion. We further showed that MKRN3 has ubiquitinase activity, that this activity is reduced by MKRN3 mutations affecting the RING finger domain, and that these mutations compromised the ability of MKRN3 to repress KISS1 and TAC3 promoter activity. These results indicate that MKRN3 acts to prevent puberty initiation, at least in part, by repressing KISS1 and TAC3 transcription and that this action may involve a MKRN3-directed ubiquitination-mediated mechanism.
Ana Paula Abreu, Carlos A. Toro, Yong Bhum Song, Victor M Navarro, Martha A. Bosch, Aysegul Eren, Joy N. Liang, Rona S. Carroll, Ana Claudia Latronico, Oline K. Ronnekleiv, Carlos F Aylwin, Alejandro Lomniczi, Sergio R. Ojeda, Ursula B Kaiser
Diabetes, obesity and Alzheimer’s disease (AD) are associated with vascular complications and impaired nitric oxide (NO) production. Furthermore, increased β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1), APP and β-amyloid (Aβ) are linked with vascular disease development and raised BACE1 and Aβ accompany hyperglycemia and hyperlipidemia. However, the causal relationship between obesity and diabetes, raised Aβ and vascular dysfunction is unclear. We report that diet-induced obesity (DIO) in mice raised plasma and vascular Aβ42 that correlated with decreased NO bioavailability, endothelial dysfunction and raised blood pressure. Genetic or pharmacological reduction of BACE1 activity and Aβ42 prevented and reversed, respectively, these outcomes. In contrast, expression of human mutant APP in mice or Aβ42 infusion into control diet-fed mice to mimic obese levels impaired NO production, vascular relaxation and raised blood pressure. In humans, raised plasma Aβ42 correlated with diabetes and endothelial dysfunction. Mechanistically, higher Aβ42 reduced endothelial NO synthase (eNOS), cyclic GMP and protein kinase G (PKG) activity independently of diet whereas endothelin-1 was increased by diet and Aβ42. Lowering Aβ42 reversed the DIO deficit in the eNOS-cGMP-PKG pathway and decreased endothelin-1. Our findings suggest that BACE1 inhibitors may have therapeutic value in the treatment of vascular disease associated with diabetes.
Paul J. Meakin, Bethany M. Coull, Zofia Tuharska, Christopher McCaffery, Ioannis Akoumianakis, Charalambos Antoniades, Jane Brown, Kathryn J. Griffin, Fiona Platt, Claire H. Ozber, Nadira Y. Yuldasheva, Natallia Makava, Anna Skromna, Alan Russell Prescott, Alison D. McNeilly, Moneeza K. Siddiqui, Colin Neil Alexander Palmer, Faisel Khan, Michael LJ Ashford
Nonalcoholic fatty liver disease (NAFLD) is becoming a major health issue as obesity increases around the world. We studied the effect of a circadian locomotor output cycles kaput (CLOCK) mutant (ClkΔ19/Δ19) protein on hepatic lipid metabolism in C57Bl6 Clkwt/wt and apolipoprotein E–deficient (Apoe−/−) mice. Both ClkΔ19/Δ19 and ClkΔ19/Δ19Apoe−/− mice developed a full spectrum of liver diseases (steatosis, steatohepatitis, cirrhosis, and hepatocellular carcinoma) recognized in human NAFLD when challenged with a Western diet, lipopolysaccharide, or CoCl2. We identified induction of cluster of differentiation 36 (CD36) and hypoxia-inducible factor 1α (HIF1α) proteins as contributing factors for NAFLD. Mechanistic studies showed that wild-type CLOCK protein interacted with the E-box enhancer elements in the promoters of the proline hydroxylase domain (PHD) proteins to increase expression. In ClkΔ19/Δ19 mice, PHD levels were low, and HIF1α protein levels were increased. When its levels were high, HIF1α interacted with the Cd36 promoter to augment expression and enhance fatty acid uptake. Thus, these studies establish a novel regulatory link among circadian rhythms, hypoxia response, fatty acid uptake, and NAFLD. The mouse models described here may be useful for further mechanistic studies in the progression of liver diseases and in the discovery of drugs for the treatment of these disorders.
Xiaoyue Pan, Joyce Queiroz, M. Mahmood Hussain
Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers and is highly resistant to current treatments. ESCC harbors a subpopulation of cells exhibiting cancer stem-like cell (CSC) properties that contribute to therapeutic resistance including radioresistance, but the molecular mechanisms in ESCC CSCs are currently unknown. Here, we report that ribosomal S6 protein kinase 4 (RSK4) plays a pivotal role in promoting CSC properties and radioresistance in ESCC. RSK4 was highly expressed in ESCC CSCs and associated with radioresistance and poor survival in ESCC patients. RSK4 was found to be a direct downstream transcriptional target of ΔNp63α, the main p63 isoform, which is frequently amplified in ESCC. RSK4 activated the β-catenin signaling pathway through direct phosphorylation of GSK-3β Ser9. Pharmacologic inhibition of RSK4 effectively reduced CSC properties and improves radiosensitivity in both nude mice and patient-derived xenograft models. Collectively, our results strongly suggest that the ΔNp63α-RSK4-GSK-3β axis plays a key role in driving CSC properties and radioresistance in ESCC, indicating that RSK4 is a promising therapeutic target for ESCC treatment.
Mingyang Li, Linni Fan, Donghui Han, Zhou Yu, Jing Ma, Yixiong Liu, Peifeng Li, Danhui Zhao, Jia Chai, Lei Jiang, Shiliang Li, Juanjuan Xiao, Qiuhong Duan, Jing Ye, Mei Shi, Yongzhan Nie, Kai-Chun Wu, Dezhong Joshua Liao, Yu Shi, Yan Wang, Qingguo Yan, Shuangping Guo, Xiu-Wu Bian, Feng Zhu, Jian Zhang, Zhe Wang
This Viewpoint calls on investigators that are developing and testing therapeutic and prophylactic approaches for COVID-19 to design studies that are inclusive of male-female differences.
Evelyne Bischof, Jeannette Wolfe, Sabra L. Klein
Haploinsufficiency of factors governing genome stability underlies hereditary breast and ovarian cancer. Homologous recombination (HR) repair is a major pathway disabled in these cancers. With the aim of identifying new candidate genes, we examined early onset breast cancer patients negative for BRCA1 and BRCA2 pathogenic variants. Here, we focused on CtIP (RBBP8 gene) that mediates HR repair through the end-resection of DNA double-strand breaks (DSB). Notably, the patients exhibited a number of rare germline RBBP8 variants, and functional analysis revealed that these variants did not affect DNA DSB end-resection efficiency. However, expression of a subset of variants led to deleterious nucleolytic degradation of stalled DNA replication forks in a manner similar to cells lacking BRCA1 or BRCA2. In contrast to BRCA1 and BRCA2, CtIP deficiency promoted the helicase-driven destabilization of RAD51 nucleofilaments at damaged DNA replication forks. Taken together, our work identifies CtIP as a critical regulator of DNA replication fork integrity, which when compromised, may predispose to the development of early onset breast cancer.
Reihaneh Zarrizi, Martin R. Higgs, Karolin Voßgröne, Maria Rossing, Birgitte Bertelsen, Muthiah Bose, Arne N. Kousholt, Heike I. Rösner, Bent Ejlertsen, Grant S. Stewart, Finn Cilius Nielsen, Claus Sørensen
Several missense mutations in the orphan transporter FLVCR2 have been reported in Fowler syndrome. Affected subjects exhibit signs of severe neurological defects. We identified the mouse ortholog Mfsd7c as a gene, which is expressed in the blood brain barrier. Here, we report the characterizations of Mfsd7c knockout (KO) mice and compare it to phenotypic findings in humans with bi-allelic FLVCR2 mutations. Global KO of Mfsd7c in mice resulted in late gestation lethality, likely due to central nervous system (CNS) phenotypes. We found that the angiogenic growth of CNS blood vessels in the brain of Mfsd7c KO embryos was inhibited in cortical ventricular zones and ganglionic eminences. Vascular tips were dilated and fused resulting in glomeruloid vessels. Nonetheless, CNS blood vessels were intact without haemorrhage. Both embryos and humans with bi-allelic FLVCR2 mutations exhibited reduced cerebral cortical layers, enlargement of the cerebral ventricles, and microcephaly. Transcriptomic analysis of Mfsd7c knockout (KO) embryonic brains revealed upregulation of genes involved in glycolysis and angiogenesis. The Mfsd7c KO brain exhibited hypoxia and neuronal cell death. Our results indicate MFSD7c is required for the normal growth of CNS blood vessels and ablation of this gene results in microcephaly-associated vasculopathy in mice and humans.
Pazhanichamy Kalailingam, Kai Qi Wang, Xiu Ru Toh, Toan Q. Nguyen, Madhuvanthi Chandrakanthan, Zafrul Hasan, Clair Habib, Aharon Schif, Francesca Clementina Radio, Bruno Dallapiccola, Karin Weiss, Long N. Nguyen
Fowler syndrome is a rare autosomal recessive brain vascular disorder caused by mutation in FLVCR2 in humans. The disease occurs during a critical period of brain vascular development, is characterized by glomeruloid vasculopathy and hydrocephalus, and is almost invariably prenatally fatal. Here, we sought to gain insights into the process of brain vascularization and the pathogenesis of Fowler Syndrome by inactivating Flvcr2 in mice. We show that Flvcr2 is necessary for angiogenic sprouting in the brain, but surprisingly dispensable for maintaining the blood brain barrier. Endothelial cells lacking Flvcr2 have altered expression of angiogenic factors, fail to adopt tip-cell properties and display reduced sprouting leading to vascular malformations similar to those seen in humans with Fowler Syndrome. Brain hypo-vascularization is associated with hypoxia and tissue infarction, ultimately causing hydrocephalus and death of mutant animals. Strikingly, despite severe vascular anomalies and brain tissue infarction, the blood-brain barrier is maintained in Flvcr2 mutant mice. Our new Fowler syndrome models therefore define the pathobiology of this disease, and provide new insights into brain angiogenesis by showing uncoupling of vessel morphogenesis and blood-brain barrier formation.
Nicolas Santander, Carlos Omar Lizama, Eman Meky, Gabriel L. McKinsey, Bongnam Jung, Dean Sheppard, Christer Betsholtz, Thomas D. Arnold
The microbiome provides resistance to infection. However, mechanisms for this are poorly understood. Here we demonstrate in a murine model that colonization with the intestinal bacterium Clostridium scindens provided protection from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte-progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone-marrow from C. scindens colonized-mice into naïve-mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Because of the known ability of the Lachnoclostridia C. scindens to metabolize the bile salt cholate, we measured deoxycholate and discovered that it was increased in the sera of C. scindens colonized specific pathogen free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone (in the absence of C. scindens) increased GMPs and provided protection from amebiasis. We have discovered a mechanism by which C. scindens and the microbially-metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with Entamoeba histolytica.
Stacey L. Burgess, Jhansi L. Leslie, Md. Jashim Uddin, David Noah Oakland, Carol A. Gilchrist, G. Brett Moreau, Koji Watanabe, Mahmoud M. Saleh, Morgan Simpson, Brandon A. Thompson, David T. Auble, Stephen D. Turner, Natasa Giallourou, Jonathan Swann, Zhen Pu, Jennie Z. Ma, Rashidul Haque, William A. Petri, Jr.