Patients with chronic inflammatory disorders such as psoriasis have an increased risk of cardiovascular disease and elevated levels of LL37, a cathelicidin host defense peptide that has both antimicrobial and proinflammatory properties. To explore if LL37 could contribute to the risk of heart disease, we examined its effects on lipoprotein metabolism and show that LL37 enhances LDL uptake in macrophages through LDLR, SR-B1 and CD36. This interaction led to increased cytosolic cholesterol in macrophages and changes in expression of lipid metabolism genes consistent with increased cholesterol uptake. Structure-function analysis and synchrotron small angle X-ray scattering show structural determinants of the LL37-LDL complex that underlie its ability to bind its receptors and promote uptake. This function of LDL uptake is unique to cathelicidins from humans and some primates and was not observed with cathelicidins from mice or rabbits. Notably, Apoe-/- mice expressing LL37 develop larger atheroma plaques than control mice and a positive correlation between plasma LL37 and OxPL-apoB levels was observed in human subjects with cardiovascular disease. These findings provide evidence that LDL uptake can be increased via interaction with LL37 and may explain the increased risk of cardiovascular disease associated with the chronic inflammatory disorders.
Yoshiyuki Nakamura, Nikhil N. Kulkarni, Toshiya Takahashi, Haleh Alimohamadi, Tatsuya Dokoshi, Edward L. Liu, Michael Shia, Tomofumi Numata, Elizabeth W.C. Luo, Adrian F. Gombart, Xiaohong Yang, Patrick Secrest, Philip L.S.M. Gordts, Sotirios Tsimikas, Gerard C.L. Wong, Richard L. Gallo
Effective immunity requires a large, diverse naïve T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we addressed how S1P enables T cell survival, and the implications for patients treated with S1PR1 antagonists. We found that S1PR1 limited apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization were required to prevent this pro-apoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naïve T cells limited B cell responses. Our findings highlighted an effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggested both limitations and additional uses of this important class of drugs.
Dhaval Dixit, Victoria M. Hallisey, Ethan Y.S. Zhu, Martyna Okuniewska, Ken Cadwell, Jerry E. Chipuk, Jordan E. Axelrad, Susan R. Schwab
The mechanisms behind a lack of efficient fear extinction in some individuals are unclear. Here, by employing a principal components analysis (PCA)-based approach, we differentiated the mice into extinction-resistant and susceptible groups. We identified that elevated synapsin 2a (Syn2a) in the infralimbic cortex (IL) to basolateral amygdala (BLA) circuit disrupted presynaptic orchestration, leading to an excitatory/inhibitory imbalance in the BLA region and causing extinction resistance. Overexpression or silencing of Syn2a levels in IL neurons replicated or alleviated behavioral, electrophysiological, and biochemical phenotypes in resistant mice. We further identified the proline-rich domain H in the C-terminal of Syn2a was indispensable for the interaction with synaptogyrin-3 (Syngr3) and demonstrated that disrupting this interaction restored extinction impairments. Molecular docking revealed ritonavir, an FDA-approved HIV drug, could disrupt Syn2a-Syngr3 binding and rescue fear extinction behavior in Syn2a-elevated mice. In summary, aberrant Syn2a elevation and its interaction with Syngr3 at the presynaptic site were crucial in fear extinction resistance, suggesting a potential therapeutic avenue for related disorders.
Xi-Ya Shen, Juan Zhang, He-Zhou Huang, Shao-Dan Li, Ling Zhou, Shi-Ping Wu, Cheng Tang, Xian Huang, Zhi-Qiang Liu, Zi-Yuan Guo, Xiang Li, Heng-Ye Man, You-Ming Lu, Ling-Qiang Zhu, Dan Liu
Mutations in the N-terminal WD40 domain of coatomer protein complex subunit α (COPA) cause a type I interferonopathy, typically characterized by alveolar hemorrhage, arthritis and nephritis. We described three heterozygous mutations in the C-terminal domain (CTD) of COPA (p.C1013S, p.R1058C and p.R1142X) in six children from three unrelated families with a similar syndrome of autoinflammation and autoimmunity. We showed that these CTD COPA mutations disrupt the integrity and the function of the coat protein complex I (COPI). In COPAR1142X and COPAR1058C fibroblasts we demonstrated that COPI dysfunction causes both an anterograde ER-to-Golgi and a retrograde Golgi-to-ER trafficking defect. The disturbed intracellular trafficking resulted in a cGAS/STING-dependent upregulation of the type I IFN signaling in patients and patient-derived cell lines, albeit through a distinct molecular mechanism in comparison to mutations in the WD40 domain of COPA. We showed that CTD COPA mutations induce an activation of the ER stress and NF-κB signaling in patient-derived primary cell lines. These results demonstrate the importance of the integrity of the CTD of COPA for COPI function and homeostatic intracellular trafficking, essential to ER homeostasis. CTD COPA mutations result in disease by increased ER stress, disturbed intracellular transport and increased pro-inflammatory signaling.
Selket Delafontaine, Alberto Iannuzzo, Tarin M. Bigley, Bram Mylemans, Ruchit R. Rana, Pieter Baatsen, M. Cecilia Poli, Daisy Rymen, Katrien Jansen, Djalila Mekahli, Ingele Casteels, Catherine Cassiman, Philippe Demaerel, Alice Lepelley, Marie-Louise Frémond, Rik Schrijvers, Xavier Bossuyt, Katlijn Vints, Wim Huybrechts, Rachida Tacine, Karen Willekens, Anniek Corveleyn, Bram Boeckx, Marco Baggio, Lisa Ehlers, Sebastian Munck, Diether Lambrechts, Arnout R.D. Voet, Leen Moens, Giorgia Bucciol, Megan A. Cooper, Carla M. Davis, Jérôme Delon, Isabelle Meyts
Aster proteins mediate the nonvesicular transport of cholesterol from the plasma membrane (PM) to the endoplasmic reticulum (ER). However, the importance of nonvesicular sterol movement for physiology and pathophysiology in various tissues is incompletely understood. Here we show that loss of Aster-B leads to diet-induced obesity and insulin resistance in female but not male mice, and that this sex difference is abolished by ovariectomy. We further demonstrate that Aster-B deficiency impairs nonvesicular cholesterol transport from the PM to the ER in ovaries in vivo, leading to hypogonadism and reduced estradiol synthesis. Female Aster-B-deficient mice exhibit reduced locomotor activity and energy expenditure, consistent with established effects of estrogens on systemic metabolism. Administration of exogenous estradiol ameliorates the diet-induced obesity phenotype of Aster-B-deficient female mice. These findings highlight the key role of Aster-B-dependent nonvesicular cholesterol transport in regulating estradiol production and protecting females from obesity.
Xu Xiao, John Paul Kennelly, An-Chieh Feng, Lijing Cheng, Beatriz Romartinez-Alonso, Alexander H. Bedard, Yajing Gao, Liujuan Cui, Stephen G. Young, John W.R. Schwabe, Peter Tontonoz
Blood vessels are continually exposed to circulating lipids and elevations of ApoB containing lipoproteins cause atherosclerosis. Lipoprotein metabolism is highly regulated by lipolysis, largely at the level of the capillary endothelium lining metabolically active tissues. How large blood vessels, the site of atherosclerotic vascular disease, regulate the flux of fatty acids (FA) into triglyceride (TG) rich lipid droplets (LD) is not known. In this study, we showed that deletion of the enzyme, adipose triglyceride lipase (ATGL) in the endothelium, led to neutral lipid accumulation in vessels and impaired endothelial dependent vascular tone and nitric oxide synthesis to promote endothelial dysfunction. Mechanistically, the loss of ATGL led to endoplasmic reticulum stress-induced inflammation in the endothelium. Consistent with this mechanism, deletion of endothelial ATGL markedly increased lesion size in a model of atherosclerosis. Together, these data demonstrate that the dynamics of FA flux through LD impacts endothelial cell homeostasis and consequently large vessel function during normal physiology and in a chronic disease state.
Nabil E. Boutagy, Ana Gamez-Mendez, Joseph W.M. Fowler, Hanming Zhang, Bal K. Chaube, Enric Esplugues, Sungwoon Lee, Daiki Horikami, Jiasheng Zhang, Kathryn M. Citrin, Abhishek K. Singh, Brian G. Coon, Yajaira Suarez, Carlos Fernandez-Hernando, William C. Sessa
Aneuploidy, a deviation from the normal chromosome copy number, is common in human embryos and is considered a primary cause of implantation failure and early pregnancy loss. Meiotic errors lead to uniformly abnormal karyotypes, while mitotic errors lead to chromosomal mosaicism: the presence of cells with at least two different karyotypes within an embryo. Knowledge about mosaicism in blastocysts mainly derives from bulk DNA sequencing of multicellular trophectoderm (TE) and/or inner cell mass (ICM) samples. However, this can only detect an average net gain or loss of DNA above a detection threshold of 20-30%. To accurately assess mosaicism, we separated the TE and ICM of 55 good quality surplus blastocysts and successfully applied single-cell whole genome sequencing (scKaryo-seq) on 1057 cells. Mosaicism involving numerical and structural chromosome abnormalities was detected in 82% of the embryos, where most abnormalities affected less than 20% of the cells. Structural abnormalities, potentially caused by replication stress and DNA damage, were observed in 69% of the embryos. In conclusion, our findings indicated that mosaicism is prevalent in good-quality blastocysts, while these blastocysts would likely be identified as normal with current bulk DNA sequencing techniques used for preimplantation genetic testing for aneuploidy (PGT-A).
Effrosyni A. Chavli, Sjoerd J. Klaasen, Diane Van Opstal, Joop S.E. Laven, Geert J.P.L. Kops, Esther B. Baart
BACKGROUND. Improving and predicting tumor response to immunotherapy remains challenging. Combination therapy with a transforming growth factor-β receptor (TGF-βR) inhibitor that targets cancer associated fibroblasts (CAFs) is promising to enhance efficacy of immunotherapies. However, the effect of this approach in clinical trials is limited, requiring in vivo methods to better assess tumor responses to combination therapy. METHODS. We measure CAFs in vivo using 68Ga-labeled fibroblast activation protein inhibitor (68Ga-FAPI)-04 for PET/CT imaging to guide combination of TGF-β inhibition and immunotherapy. 131 patients with metastatic colorectal cancer (CRC) underwent 68Ga-FAPI and 18F-fludeoxyglucose (18F-FDG) PET/CT imaging. Relationship between uptake of 68Ga-FAPI and tumor immunity was analyzed in patients. Mouse cohorts of metastatic CRC were treated with TGF-βR inhibitor combined with KN046 which blocks PD-L1 and CTLA4, followed with 68Ga-FAPI and 18F-FDG micro-PET/CT imaging to assess tumor responses. RESULTS. Patients with metastatic CRC demonstrated high uptakes of 68Ga-FAPI, along with suppressive tumor immunity and poor prognosis. TGF-βR inhibitor enhanced tumor infiltrating T cells and significantly sensitized metastatic CRC to KN046. 68Ga-FAPI PET/CT imaging accurately monitored the dynamical changes of CAFs and tumor response to combined TGF-βR inhibitor with immunotherapy. CONCLUSION. 68Ga-FAPI PET/CT imaging is powerful in assessing tumor immunity and response to immunotherapy in metastatic CRC. This study supports future clinical application of 68Ga-FAPI PET/CT to guide CRC patients for precise TGF-β inhibition plus immunotherapy, recommending 68Ga-FAPI and 18F-FDG dual PET/CT for CRC management. TRIAL REGISTRATION. CFFSTS Trial, ChiCTR2100053984, Chinese Clinical Trial Registry. FUNDING. National Natural Science Foundation of China (82072695, 32270767, 82272035,81972260).
Ke Li, Wei Liu, Hang Yu, Jiwei Chen, Wenxuan Tang, Jianpeng Wang, Ming Qi, Yuyun Sun, Xiaoping Xu, Ji Zhang, Xinxiang Li, Weijian Guo, Xiaoling Li, Shaoli Song, Shuang Tang
Melanocortin 4 receptor (MC4R) mutations are the most common cause of human monogenic obesity and are associated with hyperphagia and increased linear growth. While MC4R is known to activate Gsα/cAMP signaling, a significant proportion of obesity-associated MC4R mutations do not affect MC4R/Gsα signaling. To further explore the role of specific MC4R signaling pathways in regulation of energy balance, we examined the signaling properties of one such mutant MC4R (F51L), as well as the metabolic consequences of MC4RF51L mutation in mice. The MC4RF51L mutation produced a specific defect in MC4R/Gq/11α signaling and led to obesity, hyperphagia and increased linear growth in mice. The ability of a melanocortin agonist to acutely inhibit food intake when delivered to the paraventricular nucleus (PVN) was lost in MC4RF51L mice, as well as in wild-type mice in which a specific Gq/11α inhibitor was delivered to the PVN, providing evidence that a Gsα-independent signaling pathway, namely Gq/11α, significantly contributes to the actions of MC4R on food intake and linear growth. These results suggest that a biased MC4R agonist that primarily activates Gq/11α may be a potential agent to treat obesity with less untoward cardiovascular and other side effects.
Peter J. Metzger, Aileen Zhang, Bradley A. Carlson, Hui Sun, Zhenzhong Cui, Yongqi Li, Marshal T. Jahnke, Daniel R. Layton, Meenakshi B. Gupta, Naili Liu, Evi Kostenis, Oksana Gavrilova, Min Chen, Lee S. Weinstein
Metastasized colorectal cancer (CRC) is associated with a poor prognosis and rapid disease progression. Besides hepatic metastasis, peritoneal carcinomatosis is the major cause of death in UICC (Union for International Cancer Control) stage IV CRC patients. Insights into differential site-specific reconstitution of tumour cells and the corresponding tumour microenvironment are still missing. Here, we analysed the transcriptome of single cells derived from murine multivisceral CRC and delineated the inter-metastatic cellular heterogeneity regarding tumour epithelium, stroma and immune cells. Interestingly, we found an intercellular site-specific network of cancer associated fibroblasts and tumour epithelium during peritoneal metastasis as well as an autologous feed-forward loop in cancer stem cells. We furthermore deciphered a metastatic dysfunctional adaptive immunity by a loss of B cell dependent antigen presentation and consecutive effector T cell exhaustion. Furthermore, we demonstrated major similarities of this murine metastatic CRC model with human disease and -based on the results of our analysis- provided an auspicious site-specific immune modulatory treatment approach for stage IV CRC by intraperitoneal checkpoint inhibition.
Christopher Berlin, Bernhard Mauerer, Pierre Cauchy, Jost Luenstedt, Roman Sankowski, Lisa Marx, Reinhild Feuerstein, Luisa Schäfer, Florian R. Greten, Marina Pesic, Olaf Groß, Marco Prinz, Naomi Rühl, Laura Miketiuk, Dominik Jauch, Claudia Laessle, Andreas Jud, Esther A. Biesel, Hannes P. Neeff, Stefan Fichtner-Feigl, Philipp A. Holzner, Rebecca Kesselring
Lymphangioleiomyomatosis (LAM) is a progressive cystic lung disease caused by tuberous sclerosis complex 1/2 (TSC1/2) gene mutations in pulmonary mesenchymal cells resulting in activation of the mechanistic target of rapamycin complex 1 (mTORC1). A subset of LAM patients develops pulmonary vascular remodeling and pulmonary hypertension. Little, however, is known regarding how LAM cells communicate with endothelial cells (ECs) to trigger vascular remodeling. In end-stage LAM lung explants, we identified endothelial cell dysfunction characterized by increased proliferation, migration, defective angiogenesis, and dysmorphic endothelial tube network formation. To model LAM disease, we utilized an mTORC1 gain-of-function mouse model with a Tsc2 knock-out (Tsc2KO) specific to lung mesenchyme (Tbx4LME-CreTsc2fl/fl), similar to the mesenchyme specific genetic alterations seen in human disease. As early as 8 weeks of age, ECs from Tbx4LME-CreTsc2fl/fl mice exhibited marked transcriptomic changes despite absence of morphological changes to the distal lung microvasculature. In contrast, 1 year old Tbx4LME-CreTsc2fl/fl mice spontaneously developed pulmonary vascular remodeling with increased medial thickness. Single cell RNA-sequencing of 1 year old mouse lung identified paracrine ligands originating from Tsc2KO mesenchyme which can signal through receptors in arterial ECs. These ECs had transcriptionally altered genes including those in pathways associated with blood vessel remodeling. The proposed pathophysiologic mesenchymal ligand/ EC receptor crosstalk highlights the importance of an altered mesenchymal-EC axis in LAM and other hyperactive mTORC1-driven diseases. Since ECs in LAM patients and in Tbx4LME-CreTsc2fl/fl mice do not harbor TSC2 mutations, our study demonstrates that constitutively active mTORC1 lung mesenchymal cells orchestrate dysfunctional EC responses which contribute to pulmonary vascular remodeling.
Susan M. Lin, Ryan Rue, Alexander R. Mukhitov, Akansha Goel, Maria C. Basil, Kseniya Obraztsova, Apoorva Babu, Slaven Crnkovic, Owen Ledwell, Laura T. Ferguson, Joseph D. Planer, Ana N. Nottingham, Kanth Swaroop Vanka, Carly J. Smith, Edward Cantu III, Grazyna Kwapiszewska, Edward E. Morrisey, Jillian F. Evans, Vera P. Krymskaya
Pulmonary surfactant is a lipoprotein complex lining the alveolar surface to decrease the surface tension and facilitate inspiration. Surfactant deficiency is often seen in premature infants and also children and adults with respiratory distress syndrome. Mechanical stretch of alveolar type 2 epithelial (AT2) cells during lung expansion is the primary physiological factor that stimulates surfactant secretion; however, it is unclear whether there is a mechanosensor dedicated for this process. Here we show that loss of mechanosensitive channels TMEM63A and TMEM63B resulted in atelectasis and respiratory failure in mice due to deficit of surfactant secretion. TMEM63A/B were predominantly localized at the limiting membrane of lamellar body, a lysosome-related organelle that stores pulmonary surfactant and ATP in AT2 cells. Activation of TMEM63A/B channels during cell stretch facilitated release of surfactant and ATP from lamellar bodies fused with the plasma membrane. The released ATP evoked Ca2+ signaling in AT2 cells and potentiated exocytic fusion of more lamellar bodies. Our study uncovered a vital physiological function of TMEM63 mechanosensitive channels, which makes the lung ready for the first breath at birth and maintains respiration through the life.
Gui-Lan Chen, Jing-Yi Li, Xin Chen, Jia-Wei Liu, Qian Zhang, Jie-Yu Liu, Jing Wen, Na Wang, Ming Lei, Jun-Peng Wei, Li Yi, Jia-Jia Li, Yu-Peng Ling, He-Qiang Yi, Zhenying Hu, Jingjing Duan, Jin Zhang, Bo Zeng
In a structure-function study of sulfatides, that typically stimulate type II NKT cells, we made an unexpected discovery. We compared analogues with sphingosine or phytosphingosine chains and 24-carbon acyl chains with 0-1-2 double bonds (C or pC24:0, 24:1, or 24:2). C24:1 and C24:2 sulfatide presented by CD1d monomer on plastic stimulated type II, not type I, NKT-cell hybridomas as expected. Unexpectedly, when presented by bone-marrow-derived DCs (BMDCs), C24:2 reversed specificity to stimulate type I, not type II, NKT-cell hybridomas, mimicking the corresponding βGalCer without sulfate. It induced IFNγ-dependent immunoprotection against CT26 colon-cancer lung metastases, skewed the cytokine profile, and activated cDC1s. This was abrogated by blocking lysosomal processing with bafilomycin A1, or sulfite-blocking or deletion of arylsulfatase A that cleaves off sulfate. Thus, C24:2 is unexpectedly processed in BMDCs from a type II to a type I NKT cell-stimulating ligand, promoting tumor immunity. We believe this is the first discovery of antigen processing of glycosylceramides altering the specificity for the target cell that reverses its function from stimulating type II to stimulating type I NKT cells, introducing protective functional activity in cancer. It also uncovers a new role for antigen processing, not to allow MHC loading but to alter the cell responding.
Kumiko Nishio, Lise Pasquet, Kaddy Camara, Julia DiSapio, Shingo Kato, Kevin S. Hsu, Anja Bloom, Stewart K. Richardson, Joshua A. Welsh, Tianbo Jiang, Jennifer C. Jones, Susanna Cardell, Hiroshi Watarai, Masaki Terabe, Purevdorj B. Olkhanud, Amy R. Howell, Jay A. Berzofsky
Manale El Kharbili, Sarah K. Sasse, Lynn Sanford, Sean Jacobson, Katja Aviszus, Arnav Gupta, Claire J. Guo, Susan M. Majka, Robin D. Dowell, Anthony N. Gerber, Russell P. Bowler, Fabienne Gally
Although chronic low-grade inflammation does not cause immediate clinical symptoms, over longer term can enhance other insults or of age-dependent damage to organ systems and thereby contribute to age-related disorders, such as respiratory disorders, heart disease, metabolic disorders, autoimmunity, and cancer. However, the molecular mechanisms governing low-level inflammation are largely unknown. We discovered that Bik-deficiency causes low level inflammation even at baseline and the development of spontaneous emphysema in female but not male mice. Similarly, a single nucleotide polymorphism that reduced Bik levels was associated with increased inflammation and enhanced decline in lung function in humans. Transgenic expression of Bik in the airways of Bik-deficient mice inhibited allergen- or LPS-induced lung inflammation and reversed emphysema in female mice. Bik-deficiency increased nuclear but not cytosolic p65 levels, because Bik by modifying the BH4 domain of Bcl-2 interacted with Rpn1 and Rpn2 and enhanced proteasomal degradation of nuclear proteins. Bik-deficiency increased inflammation primarily in females because Bcl-2 and Bik levels were reduced in lung tissues and airway cells of female compared with male mice. Therefore, controlling low-grade inflammation by modifying the unappreciated role of Bik and Bcl-2 in facilitating proteasomal degradation of nuclear proteins may be crucial in treating chronic age-related diseases.
Yohannes A. Mebratu, Jane T. Jones, Congjian Liu, Zerihun H. Negasi, Mizanur Rahman, Joselyn Rojas-Quintero, George T. O'Connor, Wei Gao, Josee Dupuis, Michael H. Cho, Augusto A. Litonjua, Scott Randell, Yohannes Tesfaigzi
Altered tryptophan catabolism has been identified in inflammatory diseases like rheumatoid arthritis (RA) and spondyloarthritis (SpA), but the causal mechanisms linking tryptophan metabolites to disease are unknown. Using the collagen-induced arthritis (CIA) model we identified alterations in tryptophan metabolism, and specifically indole, that correlated with disease. We demonstrated that both bacteria and dietary tryptophan were required for disease, and indole supplementation was sufficient to induce disease in their absence. When mice with CIA on a low-tryptophan diet were supplemented with indole, we observed significant increases in serum IL-6, TNF, and IL-1β; splenic RORγt+CD4+ T cells and ex vivo collagen-stimulated IL-17 production; and a pattern of anti-collagen antibody isotype switching and glycosylation that corresponded with increased complement fixation. IL-23 neutralization reduced disease severity in indole-induced CIA. Finally, exposure of human colon lymphocytes to indole increased expression of genes involved in IL-17 signaling and plasma cell activation. Altogether, we propose a mechanism by which intestinal dysbiosis during inflammatory arthritis results in altered tryptophan catabolism, leading to indole stimulation of arthritis development. Blockade of indole generation may present a unique therapeutic pathway for RA and SpA.
Brenda J. Seymour, Brandon Trent, Brendan E. Allen, Adam J. Berlinberg, Jimmy Tangchittsumran, Widian K. Jubair, Meagan E. Chriswell, Sucai Liu, Alfredo Ornelas, Andrew Stahly, Erica E. Alexeev, Alexander S. Dowdell, Sunny L. Sneed, Sabrina Fechtner, Jennifer M. Kofonow, Charles E. Robertson, Stephanie M. Dillon, Cara C. Wilson, Robert M. Anthony, Daniel N. Frank, Sean P. Colgan, Kristine A. Kuhn
Aplasia cutis congenita (ACC) is a congenital epidermal defect of the midline scalp and has been proposed to be due to a primary keratinocyte abnormality. Why it forms mainly at this anatomic site has remained a longstanding enigma. KCTD1 mutations cause ACC, ectodermal abnormalities, and kidney fibrosis, whereas KCTD15 mutations cause ACC and cardiac outflow tract abnormalities. Here, we find that KCTD1 and KCTD15 can form multimeric complexes and can compensate for each other's loss, and that disease mutations are dominant-negative, resulting in lack of KCTD1/KCTD15 function. We demonstrate that KCTD15 is critical for cardiac outflow tract development, whereas KCTD1 regulates distal nephron function. Combined inactivation of KCTD1/KCTD15 in keratinocytes results in abnormal skin appendages, but not in ACC. Instead, KCTD1/KCTD15 inactivation in neural crest cells results in ACC linked to midline skull defects, demonstrating that ACC is not caused by a primary defect in keratinocytes but is a secondary consequence of impaired cranial neural crest cells giving rise to midline cranial suture cells that express keratinocyte-promoting growth factors. Our findings explain the clinical observations in patients with KCTD1 versus KCTD15 mutations, establish KCTD1/KCTD15 as critical regulators of ectodermal and neural crest cell functions, and define ACC as a neurocristopathy.
Jackelyn R. Raymundo, Hui Zhang, Giovanni Smaldone, Wenjuan Zhu, Kathleen E. Daly, Benjamin J. Glennon, Giovanni Pecoraro, Marco Salvatore, William A. Devine, Cecilia W. Lo, Luigi Vitagliano, Alexander G. Marneros
Itaconate has emerged as a critical immunoregulatory metabolite. Here, we examined the therapeutic potential of itaconate in atherosclerosis. We found that both itaconate and the enzyme that synthesizes it, aconitate decarboxylase 1 (Acod1, also known as “immune-responsive gene 1”/IRG1) are upregulated during atherogenesis in mice. Deletion of Acod1 in myeloid cells exacerbated inflammation and atherosclerosis in vivo and resulted in an elevated frequency of a specific subset of M1-polarized proinflammatory macrophages in the atherosclerotic aorta. Importantly, Acod1 levels were inversely correlated with clinical occlusion in atherosclerotic human aorta specimens. Treating mice with the itaconate derivative 4-ocytyl itaconate attenuated inflammation and atherosclerosis induced by high cholesterol. Mechanistically, we found that the antioxidant transcription factor, Nuclear factor erythroid-2 Related Factor 2 (Nrf2) was required for itaconate to suppress macrophage activation induced by oxidized lipids in vitro and to decrease atherosclerotic lesion areas in vivo. Overall, our work shows that itaconate suppresses atherogenesis by inducing Nrf2-dependent inhibition of proinflammatory responses in macrophages. Activation of the itaconate pathway may represent an important approach to treat atherosclerosis.
Jianrui Song, Yanling Zhang, Ryan A. Frieler, Anthony Andren, Sherri C. Wood, Daniel J. Tyrrell, Peter Sajjakulnukit, Jane C. Deng, Costas A. Lyssiotis, Richard M. Mortensen, Morgan Salmon, Daniel R. Goldstein
Several canonical translocations produce oncofusion genes that can initiate Acute Myeloid Leukemia (AML). Although each translocation is associated with unique features, the mechanisms responsible remain unclear. While proteins interacting with each oncofusion are known to be relevant for how they act, these interactions have not yet been systematically defined. To address this issue in an unbiased fashion, we fused a promiscuous biotin ligase ("TurboID") in-frame with three favorable-risk acute myeloid leukemia (AML) oncofusion cDNAs (PML::RARA, RUNX1::RUNX1T1, and CBFB::MYH11), and identified their interacting proteins in primary murine hematopoietic cells. The PML::RARA- and RUNX1::RUNX1T1-TurboID fusion proteins labeled common and unique nuclear repressor complexes, implying their nuclear localization. However, CBFB::MYH11-TurboID interacting proteins were largely cytoplasmic, probably due to an interaction of the MYH11 domain with several cytoplasmic myosin-related proteins. Using a variety of methods, we showed that the CBFB domain of CBFB::MYH11 sequesters RUNX1 in cytoplasmic aggregates; these findings were confirmed in primary human AML cells. Paradoxically, CBFB::MYH11 expression was associated with increased RUNX1/2 expression, suggesting the presence of a sensor for reduced functional RUNX1 protein, and a feedback loop that that may attempt to compensate by increasing RUNX1/2 transcription. These findings may have broad implications for AML pathogenesis.
Ryan B. Day, Julia A. Hickman, Ziheng Xu, Casey D.S. Katerndahl, Francesca Ferraro, Sai Mukund Ramakrishnan, Petra Erdmann-Gilmore, Robert W. Sprung, Yiling Mi, R. Reid Townsend, Christopher A. Miller, Timothy J. Ley
Non-alcoholic fatty liver disease (NAFLD) is prevalent in the majority of obese individuals, but in a subset, this progresses to non-alcoholic steatohepatitis (NASH) and fibrosis. The mechanisms that prevent NASH and fibrosis in the majority of NAFLD patients remain unclear. Here we report that NAD(P)H oxidase (NOX)-4 and nuclear factor erythroid 2-related factor 2 (NFE2L2) were elevated in hepatocytes early in disease progression to prevent NASH/fibrosis. Mitochondrial-derived reactive oxygen species (ROS) activated NFE2L2 to induce the expression of NOX4, which in turn generated H2O2 to exacerbate the NFE2L2 antioxidant defense response. The deletion or inhibition of NOX4 in hepatocytes decreased ROS and attenuated antioxidant defense to promote mitochondrial oxidative stress, damage proteins and lipids, diminish insulin signalling and promote cell death upon oxidant challenge. Hepatocyte NOX4 deletion in high fat fed obese mice, which otherwise develop steatosis, but not NASH, resulted in hepatic oxidative damage, inflammation and T cell recruitment to drive NASH and fibrosis, whereas NOX4 overexpression tempered the development of NASH/fibrosis in mice fed a NASH-promoting diet. Thus, mitochondrial- and NOX4-derived ROS function in concert to drive a NFE2L2 antioxidant defense response to attenuate oxidative liver damage and the progression to NASH/fibrosis in obesity.
Spencer Greatorex, Supreet Kaur, Chrysovalantou E. Xirouchaki, Pei Kee Goh, Florian Wiede, Amanda J. Genders, Melanie Tran, YaoYao Jia, Arthe Raajendiran, Wendy A. Brown, Catriona A. McLean, Junichi Sadoshima, Matthew J. Watt, Tony Tiganis