Axon loss contributes to many common neurodegenerative disorders. In healthy axons, the axon survival factor NMNAT2 inhibits SARM1, the central executioner of programmed axon degeneration. We identified two rare NMNAT2 missense variants in two brothers afflicted with a progressive neuropathy syndrome. The polymorphisms resulted in amino acid substitutions, V98M and R232Q, which reduced NMNAT2 NAD+-synthetase activity. We generated a mouse model of the human syndrome and found that Nmnat2V98M/Nmnat2R232Q compound-heterozygous CRISPR mice survived to adulthood but developed progressive motor dysfunction, peripheral axon loss, and macrophage infiltration. These disease phenotypes were all SARM1-dependent. Remarkably, macrophage depletion therapy blocked and reversed neuropathic phenotypes in Nmnat2V98M/R232Q mice, identifying a SARM1-dependent neuroimmune mechanism as a key driver of disease pathogenesis. These findings demonstrate that SARM1 induces an inflammatory neuropathy and highlight the potential of immune therapy to treat this rare syndrome and other neurodegenerative conditions associated with NMNAT2 loss and SARM1 activation.
Caitlin B. Dingwall, Amy Strickland, Sabrina W. Yum, Aldrin Kay-Yuen Yim, Jian Zhu, Peter L. Wang, Yurie Yamada, Robert E. Schmidt, Yo Sasaki, A. Joseph Bloom, Aaron DiAntonio, Jeffrey Milbrandt
Multiple genetic loci have been reported for progeroid syndromes. However, the molecular defects in some extremely rare forms of progeria have yet to be elucidated. Here we report a 21-year-old man of Chinese origin who had a novel autosomal recessive form of progeria, characterized by severe dwarfism, mandibular hypoplasia, hyperopia and partial lipodystrophy. Analyses of exome sequencing data of the entire family revealed only one rare homozygous missense variant, (c.86C>T; p.Pro29Leu), in TOMM7 in the proband, while the parents and two unaffected siblings were heterozygous for the variant. TOMM7, a nuclear gene, encodes a translocase in the outer mitochondrial membrane. The TOMM complex constitutes the outer membrane pore for import of several preproteins into mitochondria. Proteomics analyses of mitochondria from cultured fibroblasts of the proband, as compared to control fibroblasts, revealed increases in several proteins involved in oxidative phosphorylation, but reduced abundance of proteins involved in the phospholipid metabolism. We also observed elevated basal and maximal oxygen consumption rates in the fibroblasts from the proband as compared to control fibroblasts. We conclude that altered mitochondrial protein import due to loss of function bi-allelic variant in TOMM7 can cause severe growth retardation and progeroid features.
Abhimanyu Garg, Wee-Teik Keng, Zhenkang Chen, Adwait Amod Sathe, Chao Xing, Pavithira Devi Kailasam, Yanqiu Shao, Nicholas P. Lesner, Claire B. Llamas, Anil K. Agarwal, Prashant Mishra
As a highly regenerative organ, the intestine is a promising source for cellular reprogramming to replace lost pancreatic β-cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by FoxO1 ablation, but their numbers are limited. In this study we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage tracing experiments show that upon genetic or pharmacologic FoxO1 ablation the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate β-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGFβ that, when tested in insulin-deficient STZ or NOD diabetic animals resulted in near-normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach to replace insulin treatment in diabetes.
Wen Du, Junqiang Wang, Taiyi Kuo, Liheng Wang, Wendy M. McKimpson, Jinsook Son, Hitoshi Watanabe, Takumi Kitamoto, Yun-Kyoung Lee, Remi J. Creusot, Lloyd E. Ratner, Kasi McCune, Ya-Wen Chen, Brendan H. Grubbs, Matthew E. Thornton, Jason Fan, Nishat Sultana, Bryan S. Diaz, Iyshwarya Balasubramanian, Nan Gao, Sandro Belvedere, Domenico Accili
The role of natural killer (NK) cells against HIV-1 infections remains to be elucidated in vivo. While humanized mouse models potentially could be used to directly evaluate humans NK cell responses during HIV-1 infection, improved functional development of human NK cells in these hosts is needed. Here we report the humanized MISTRG-6-15 mouse model, in which NK cells were quick to expand and exhibit degranulation, cytotoxicity, and pro-inflammatory cytokine production in non-lymphoid organs upon HIV-1 infection, but had reduced functionality in lymphoid organs. Although HIV-1 infection induced functional impairment of NK cells, antiretroviral therapy reinvigorated NK cells in response to HIV-1 rebound after analytic treatment interruption. Moreover, a broadly neutralizing antibody PGT121 enhanced NK cell function in vivo, consistent with antibody-dependent cellular cytotoxicity. Monoclonal antibody depletion of NK cells resulted in higher viral loads in multiple non-lymphoid organs. Overall, our results in humanized MISTRG-6-15 mice demonstrated that NK cells provided direct anti-HIV-1 responses in vivo but were limited in their responses in lymphoid organs.
Can M. Sungur, Qiankun Wang, Ayşe N. Ozantürk, Hongbo Gao, Aaron J. Schmitz, Marina Cella, Wayne M. Yokoyama, Liang Shan
Targeting lineage-defined transcriptional dependencies has emerged as an effective therapeutic strategy in cancer treatment. Through screening for molecular vulnerabilities of mantle cell lymphoma (MCL), we identified a set of transcription factors (TFs) including FOXO1, EBF1, PAX5, and IRF4 that are essential for MCL propagation. Integrated chromatin immunoprecipitation and sequencing (ChIP-seq) with transcriptional network reconstruction analysis revealed FOXO1 as a master regulator that acts upstream in the regulatory TF hierarchy. FOXO1 is both necessary and sufficient to drive MCL lineage commitment through supporting the lineage-specific transcription programs. We further show that FOXO1, but not its close paralog FOXO3, can reprogram myeloid leukemia cells and induce B-lineage gene expression. Finally, we demonstrate that cpd10, a small molecule identified from an enriched FOXO1 inhibitor library, induces a robust cytotoxic response of MCL cells in vitro and suppresses MCL progression in vivo. Our findings establish FOXO1 inhibition as a therapeutic strategy targeting lineage-driven transcriptional addiction in MCL.
Ja-Young Jang, Inah Hwang, Heng Pan, Jun Yao, Lapo Alinari, Eddie Imada, Claudio Zanettini, Michael Kluk, Yizhe Wang, Yun-Kyoung Lee, Hua V. Lin, Xiangao Huang, Maurizio Di Liberto, Zhengming Chen, Karla V. Ballman, Lewis C. Cantley, Luigi Marchionni, Giorgio Inghirami, Olivier Elemento, Robert Baiocchi, Selina Chen-Kiang, Sandro Belvedere, Hongwu Zheng, Jihye Paik
BACKGROUND. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies with unpredictable responses to chemotherapy. Approaches to assay patient tumors before treatment and identify effective treatment regimens based on tumor sensitivities are lacking. We developed an organoid-based platform (OBP) to visually quantify patient derived organoid (PDO) responses to drug treatments and associated tumor-stromal modulation for personalized PDAC therapy. METHODS. We retrospectively quantified apoptotic responses and tumor-stromal cell proportions in patient-derived organoids (PDOs) via 3D immunofluorescence imaging through annexin A5, α-smooth muscle actin (α-SMA), and cytokeratin 19 (CK-19) levels. Simultaneously, an ex vivo organoid drug sensitivity assay (ODSA) was used to measure responses to standard of care (SOC) regimens. Differences between ODSA results and patient tumor responses were assessed by exact McNemar test. RESULTS. Immunofluorescent signals, organoid growth curves, and Ki-67 levels were measured and authenticated through the OBP for up to 14 days. ODSA drug responses were not different from patient tumor responses as reflected by CA19-9 reductions following neoadjuvant chemotherapy (P = 0.99). PDOs demonstrated unique apoptotic and tumor-stromal modulation profiles (P < 0.0001). α-SMA/CK-19 ratio levels > 1.0 were associated with improved outcomes (P = 0.0179), and longer parental patient survival by Kaplan-Meier analysis (P = 0.0046). CONCLUSION. Heterogenous apoptotic drug responses and tumor-stromal modulation are present in PDOs after SOC chemotherapy. Ratios of α-SMA and CK-19 levels in PDOs are associated with patient survival and the OBP could aid in the selection of personalized therapies to improve the efficacy of systemic therapy in PDAC patients.
Ya'an Kang, Jenying Deng, Jianhua Ling, Xinqun Li, Yi-Ju Chiang, Eugene J. Koay, Huamin Wang, Jared K. Burks, Paul J. Chiao, Mark W. Hurd, Manoop S. Bhutani, Jeffrey H. Lee, Brian R. Weston, Anirban Maitra, Naruhiko Ikoma, Ching-Wei D. Tzeng, Jeffrey E. Lee, Ronald A. DePinho, Robert A. Wolff, Shubham Pant, Florencia McAllister, Matthew H.G. Katz, Jason B. Fleming, Michael P. Kim
Multisystem inflammatory syndrome in children (MIS-C) evolves in some pediatric patients following acute infection with SARS-CoV-2 by hitherto unknown mechanisms. Whereas acute-COVID-19 severity and outcome were previously correlated with Notch4 expression on regulatory T (Treg) cells, here we show that the Treg cells in MIS-C are destabilized through a Notch1-dependent mechanism. Genetic analysis revealed that MIS-C patients were enriched in rare deleterious variants impacting inflammation and autoimmunity pathways, including dominant-negative mutations in the Notch1 regulators NUMB and NUMBL leading to Notch1 upregulation. Notch1 signaling in Treg cells induced CD22, leading to their destabilization in a mTORC1-dependent manner and to the promotion of systemic inflammation. These results establish a Notch1-CD22 signaling axis that disrupts Treg cell function in MIS-C and point to distinct immune checkpoints controlled by individual Treg cell Notch receptors that shape the inflammatory outcome in SARS-CoV-2 infection.
Mehdi Benamar, Qian Chen, Janet Chou, Amélie M. Julé, Rafik Boudra, Paola Contini, Elena Crestani, Peggy S. Lai, Muyun Wang, Jason Fong, Shira Rockwitz, Pui Y. Lee, Tsz Man Fion Chan, Ekin Zeynep Altun, Eda Kepenekli, Elif Karakoc-Aydiner, Ahmet Ozen, Perran Boran, Fatih Aygun, Pınar Önal, Ayse Ayzit Kilinc Sakalli, Haluk Cokugras, Metin Yusuf Gelmez, Fatma B. Oktelik, Esin Cetin Aktas, Yuelin Zhong, Maria L. Taylor, Katherine Irby, Natasha B. Halasa, Elizabeth H. Mack, Sara Signa, Ignazia Prigione, Marco Gattorno, Nicola Cotugno, Donato Amodio, Raif S. Geha, Mary Beth Son, Jane W. Newburger, Pankaj B. Agrawal, Stefano Volpi, Paolo Palma, Ayca Kiykim, Adrienne Randolph, Gunnur Deniz, Safa Baris, Raffaele De Palma, Klaus Schmitz-Abe, Louis-Marie Charbonnier, Lauren A. Henderson, Talal A. Chatila
BACKGROUND. Heterologous effects of vaccines are mediated by ‘trained immunity’ whereby myeloid cells are metabolically and epigenetically reprogrammed resulting in heightened responses to subsequent insults. Adenovirus vaccine vector has been reported to induce trained immunity in mice. Therefore, we sought to determine if the ChAdOx1 nCoV-19 vaccine (AZD1222), which uses an adenoviral vector, could induce trained immunity in vivo in humans. METHODS. Ten healthy volunteers donated blood on the day before receiving the ChAdOx1 nCoV-19 vaccine and on day 14, 56 and 90 post vaccination. Monocytes were purified from PBMC; cell phenotype was determined by flow cytometry, expression of metabolic enzymes were quantified by RT-qPCR and production of cytokines and chemokine in response to stimulation ex vivo were analyzed by multiplex ELISA. RESULTS. Monocyte frequency and count were increased in peripheral blood up to 3 months post vaccination compared with their own pre-vaccine control. Expression of HLA-DR, CD40 and CD80 was enhanced on monocytes for up to 3 months following vaccination. Moreover, monocytes had increased expression of glycolysis-associated enzymes 2 months post vaccination. Upon stimulation ex vivo with unrelated antigens, monocytes produced increased IL-1β, IL-6, IL-10, CXCL1, and MIP-1α, and decreased TNF, compared with pre-vaccine controls. Resting monocytes produced more IFN-γ, IL-18, and MCP-1 up to 3 months post vaccination compared with pre-vaccine controls. CONCLUSION. These data provide evidence for the induction of trained immunity following a single dose of the ChAdOx1 nCoV-19 vaccine. FUNDING. This work was funded by The Health Research Board (EIA-2019-010) and Science Foundation Ireland Strategic Partnership Programme (Proposal ID 20/SPP/3685).
Dearbhla M. Murphy, Donal J. Cox, Sarah A. Connolly, Eamon P Breen, Aenea A.I. Brugman, James J. Phelan, Joseph Keane, Sharee A. Basdeo
SARS-CoV-2 infection in immunocompromised individuals is associated with prolonged virus shedding and evolution of viral variants. Rapamycin and its analogs (rapalogs, including everolimus, temsirolimus, and ridaforolimus) are FDA-approved as mTOR inhibitors for the treatment of human diseases, including cancer and autoimmunity. Rapalog use is commonly associated with increased susceptibility to infection, which has been traditionally explained by impaired adaptive immunity. Here, we show that exposure to rapalogs increases susceptibility to SARS-CoV-2 infection in tissue culture and in immunologically naive rodents by antagonizing the cell-intrinsic immune response. By identifying one rapalog (ridaforolimus) that is less potent in this regard, we demonstrate that rapalogs promote Spike-mediated entry into cells by triggering the degradation of antiviral proteins IFITM2 and IFITM3 via an endolysosomal remodeling program called microautophagy. Rapalogs that increase virus entry inhibit the mTOR-mediated phosphorylation of the transcription factor TFEB, which facilitates its nuclear translocation and triggers microautophagy. In rodent models of infection, injection of rapamycin prior to and after virus exposure resulted in elevated SARS-CoV-2 replication and exacerbated viral disease, while ridaforolimus had milder effects. Overall, our findings indicate that preexisting use of certain rapalogs may elevate host susceptibility to SARS-CoV-2 infection and disease by activating lysosome-mediated suppression of intrinsic immunity.
Guoli Shi, Abhilash I. Chiramel, Tiansheng Li, Kin Kui Lai, Adam D. Kenney, Ashley Zani, Adrian C Eddy, Saliha Majdoul, Lizhi Zhang, Tirhas Dempsey, Paul A. Beare, Swagata Kar, Jonathan W. Yewdell, Sonja M Best, Jacob S. Yount, Alex A. Compton
Susanne L. Linderman, Lilin Lai, Estefany L. Bocangel Gamarra, Max S.Y. Lau, Srilatha Edupuganti, Diya Surie, Mark W. Tenforde, James D. Chappell, Nicholas M. Mohr, Kevin W. Gibbs, Jay S. Steingrub, Matthew C. Exline, Nathan I. Shapiro, Anne E. Frosch, Nida Qadir, Meredith E. Davis-Gardner, M. Juliana McElrath, Adam S. Lauring, Mehul S. Suthar, Manish M. Patel, Wesley H. Self, Rafi Ahmed
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