Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19
Grant et al. report that COVID-19 is distinguished from other causes of pneumonia by cumulative, but not peak, cytokine exposure, which is associated with inflammatory activation of microglia. The cover art is a stylized version of proinflammatory microglia, with the image rotated around a central axis to create a stellate, fractal-like pattern.
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Technical Advance
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Abstract
Evaluating the response to immune checkpoint inhibitors (ICIs) remains an unmet challenge in triple-negative breast cancer (TNBC). The requirement for cholesterol in the activation and function of T cells led us to hypothesize that quantifying cellular accumulation of this molecule could distinguish successful from ineffective checkpoint immunotherapy. To analyze accumulation of cholesterol by T cells in the immune microenvironment of breast cancer, we leveraged the PET radiotracer, eFNP-59. eFNP-59 is an analog of cholesterol that our group validated as an imaging biomarker for cholesterol uptake in preclinical models and initial human studies. In immunocompetent mouse models of TNBC, we found that elevated uptake of exogenous labeled cholesterol analogs functions as a marker for T cell activation. When comparing ICI-responsive and -nonresponsive tumors directly, uptake of fluorescent cholesterol and eFNP-59 increased in T cells from ICI-responsive tumors. We discovered that accumulation of cholesterol by T cells increased in ICI-responding tumors that received anti–PD-1 checkpoint immunotherapy. In patients with TNBC, tumors containing cycling T cells had features of cholesterol uptake and trafficking within those populations. These results suggest that uptake of exogenous cholesterol analogs by tumor-infiltrating T cells allows detection of T cell activation and has potential to assess the success of ICI therapy.
Authors
Nicholas G. Ciavattone, Nan Guan, Alex Farfel, Jenelle Stauff, Timothy Desmond, Benjamin L. Viglianti, Peter J.H. Scott, Allen F. Brooks, Gary D. Luker
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Research Articles
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Abstract
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.
Authors
Idil Apak Evans, Xingshen Sun, Bo Liang, Amber R. Vegter, Lydia Guo, Thomas J. Lynch, Yan Zhang, Yulong Zhang, Yaling Yi, Yu Yang, Zehua Feng, Soo Yeun Park, Amanita Shonka, Hannah McCumber, Lisi Qi, Peipei Wu, Guangming Liu, Allison Lacina, Kai Wang, Katherine N. Gibson-Corley, David K. Meyerholz, Dominique H. Limoli, Bradley H. Rosen, Ziying Yan, Douglas J. Bartels, John F. Engelhardt
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Skeletal muscle wasting results from numerous pathological conditions affecting both the musculoskeletal and nervous systems. A unifying feature of these pathologies is the upregulation of members of the E3 ubiquitin ligase family, resulting in increased proteolytic degradation of target proteins. Despite the critical role of E3 ubiquitin ligases in regulating muscle mass, the specific proteins they target for degradation and the mechanisms by which they regulate skeletal muscle homeostasis remain ill-defined. Here, using zebrafish loss-of-function models combined with in vivo cell biology and proteomic approaches, we reveal a role of atrogin-1 in regulating the levels of the endoplasmic reticulum chaperone BiP. Loss of atrogin-1 resulted in an accumulation of BiP, leading to impaired mitochondrial dynamics and a subsequent loss in muscle fiber integrity. We further implicated a disruption in atrogin-1–mediated BiP regulation in the pathogenesis of Duchenne muscular dystrophy. We revealed that BiP was not only upregulated in Duchenne muscular dystrophy, but its inhibition using pharmacological strategies, or by upregulating atrogin-1, significantly ameliorated pathology in a zebrafish model of Duchenne muscular dystrophy. Collectively, our data implicate atrogin-1 and BiP in the pathogenesis of Duchenne muscular dystrophy and highlight atrogin-1’s essential role in maintaining muscle homeostasis.
Authors
Avnika A. Ruparelia, Margo Montandon, Jo Merriner, Cheng Huang, Siew Fen Lisa Wong, Carmen Sonntag, Justin P. Hardee, Gordon S. Lynch, Lee B. Miles, Ashley Siegel, Thomas E. Hall, Ralf B. Schittenhelm, Peter D. Currie
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Mesenchymal stem cells (MSCs), suffering from diverse gene hits, undergo malignant transformation and aberrant osteochondral differentiation. Src homology region 2–containing protein tyrosine phosphatase 2 (SHP2), a nonreceptor protein tyrosine phosphatase, regulates multicellular differentiation, proliferation, and transformation. However, the role of SHP2 in MSC fate determination remains unclear. Here, we showed that MSCs bearing the activating SHP2E76K mutation underwent malignant transformation into sarcoma stem-like cells. We revealed that the SHP2E76K mutation in mouse MSCs led to hyperactive mitochondrial metabolism by activating mitochondrial complexes I and III. Inhibition of complexes I and III prevented hyperactive mitochondrial metabolism and malignant transformation of SHP2E76K MSCs. Mechanistically, we verified that SHP2 underwent liquid-liquid phase separation (LLPS) in SHP2E76K MSCs. SHP2 LLPS led to its dissociation from complexes I and III, causing their hyperactivation. Blockade of SHP2 LLPS by LLPS-defective mutations or allosteric inhibitors suppressed complex I and III hyperactivation as well as malignant transformation of SHP2E76K MSCs. These findings reveal that complex I and III hyperactivation driven by SHP2 LLPS promotes malignant transformation of SHP2E76K MSCs and suggest that inhibition of SHP2 LLPS could be a potential therapeutic target for the treatment of activated SHP2–associated cancers.
Authors
Chen Kan, Zhenya Tan, Liwei Liu, Bo Liu, Li Zhan, Jicheng Zhu, Xiaofei Li, Keqiong Lin, Jia Liu, Yakun Liu, Fan Yang, Mandy Wong, Siying Wang, Hong Zheng
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Glioblastoma (GBM) remains an incurable disease, requiring more effective therapies. Through interrogation of publicly available CRISPR and RNAi library screens, we identified the α-ketoglutarate dehydrogenase (OGDH) gene, which encodes an enzyme that is part of the tricarboxylic acid (TCA) cycle, as essential for GBM growth. Moreover, by combining transcriptome and metabolite screening analyses, we discovered that loss of function of OGDH by the clinically validated drug compound CPI-613 was synthetically lethal with Bcl-xL inhibition (genetically and through the clinically validated BH3 mimetic, ABT263) in patient-derived xenografts as well neurosphere GBM cultures. CPI-613–mediated energy deprivation drove an integrated stress response with an upregulation of the BH3-only domain protein, Noxa, in an ATF4-dependent manner, as demonstrated by genetic loss-of-function experiments. Consistently, silencing of Noxa attenuated cell death induced by CPI-613 in model systems of GBM. In patient-derived xenograft models of GBM in mice, the combination treatment of ABT263 and CPI-613 suppressed tumor growth and extended animal survival more potently than each compound on its own. Therefore, combined inhibition of Bcl-xL along with disruption of the TCA cycle might be a treatment strategy for GBM.
Authors
Trang T.T. Nguyen, Consuelo Torrini, Enyuan Shang, Chang Shu, Jeong-Yeon Mun, Qiuqiang Gao, Nelson Humala, Hasan O. Akman, Guoan Zhang, Mike-Andrew Westhoff, Georg Karpel-Massler, Jeffrey N. Bruce, Peter Canoll, Markus D. Siegelin
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IL-17C is an epithelial cell–derived proinflammatory cytokine whose transcriptional regulation remains unclear. Analysis of the IL17C promoter region identified TCF4 as putative regulator, and siRNA knockdown of TCF4 in human keratinocytes (KCs) increased IL17C. IL-17C stimulation of KCs (along with IL-17A and TNF-α stimulation) decreased TCF4 and increased NFKBIZ and ZC3H12A expression in an IL-17RA/RE–dependent manner, thus creating a feedback loop. ZC3H12A (MCPIP1/Regnase-1), a transcriptional immune-response regulator, also increased following TCF4 siRNA knockdown, and siRNA knockdown of ZC3H12A decreased NFKBIZ, IL1B, IL36G, CCL20, and CXCL1, revealing a proinflammatory role for ZC3H12A. Examination of lesional skin from the KC-Tie2 inflammatory dermatitis mouse model identified decreases in TCF4 protein concomitant with increases in IL-17C and Zc3h12a that reversed following the genetic elimination of Il17c, Il17ra, and Il17re and improvement in the skin phenotype. Conversely, interference with Tcf4 in KC-Tie2 mouse skin increased Il17c and exacerbated the inflammatory skin phenotype. Together, these findings identify a role for TCF4 in the negative regulation of IL-17C, which, alone and with TNF-α and IL-17A, feed back to decrease TCF4 in an IL-17RA/RE–dependent manner. This loop is further amplified by IL-17C–TCF4 autocrine regulation of ZC3H12A and IL-17C regulation of NFKBIZ to promote self-sustaining skin inflammation.
Authors
Yanyun Jiang, Dennis Gruszka, Chang Zeng, William R. Swindell, Christa Gaskill, Christian Sorensen, Whitney Brown, Roopesh Singh Gangwar, Lam C. Tsoi, Joshua Webster, Sigrún Laufey Sigurðardóttir, Mrinal K. Sarkar, Ranjitha Uppala, Austin Kidder, Xianying Xing, Olesya Plazyo, Enze Xing, Allison C. Billi, Emanual Maverakis, J. Michelle Kahlenberg, Johann E. Gudjonsson, Nicole L. Ward
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Patients with Fabry disease suffer from chronic debilitating pain and peripheral sensory neuropathy with minimal treatment options, but the cellular drivers of this pain are unknown. Here, we propose a mechanism we believe to be novel in which altered signaling between Schwann cells and sensory neurons underlies the peripheral sensory nerve dysfunction we observed in a genetic rat model of Fabry disease. Using in vivo and in vitro electrophysiological recordings, we demonstrated that Fabry rat sensory neurons exhibited pronounced hyperexcitability. Schwann cells probably contributed to this finding because application of mediators released from cultured Fabry Schwann cells induced spontaneous activity and hyperexcitability in naive sensory neurons. We examined putative algogenic mediators using proteomic analysis and found that Fabry Schwann cells released elevated levels of the protein p11 (S100A10), which induced sensory neuron hyperexcitability. Removal of p11 from Fabry Schwann cell media caused hyperpolarization of neuronal resting membrane potentials, indicating that p11 may contribute to the excessive neuronal excitability caused by Fabry Schwann cells. These findings demonstrate that sensory neurons from rats with Fabry disease exhibit hyperactivity caused in part by Schwann cell release of the protein p11.
Authors
Tyler B. Waltz, Dongman Chao, Eve K. Prodoehl, Jonathan D. Enders, Vanessa L. Ehlers, Bhavya S. Dharanikota, Nancy M. Dahms, Elena Isaeva, Quinn H. Hogan, Bin Pan, Cheryl L. Stucky
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Sepsis is a leading cause of mortality worldwide, and pneumonia is the most common cause of sepsis in humans. Low levels of high-density lipoprotein cholesterol (HDL-C) levels are associated with an increased risk of death from sepsis, and increasing levels of HDL-C by inhibition of cholesteryl ester transfer protein (CETP) decreases mortality from intraabdominal polymicrobial sepsis in APOE*3-Leiden.CETP mice. Here, we show that treatment with the CETP inhibitor (CETPi) anacetrapib reduced mortality from Streptococcus pneumoniae–induced sepsis in APOE*3-Leiden.CETP and APOA1.CETP mice. Mechanistically, CETP inhibition reduced the host proinflammatory response via attenuation of proinflammatory cytokine transcription and release. This effect was dependent on the presence of HDL, leading to attenuation of immune-mediated organ damage. In addition, CETP inhibition promoted monocyte activation in the blood prior to the onset of sepsis, resulting in accelerated macrophage recruitment to the lung and liver. In vitro experiments demonstrated that CETP inhibition significantly promoted the activation of proinflammatory signaling in peripheral blood mononuclear cells and THP1 cells in the absence of HDL; this may represent a mechanism responsible for improved bacterial clearance during sepsis. These findings provide evidence that CETP inhibition represents a potential approach to reduce mortality from pneumosepsis.
Authors
Haoyu Deng, Wan Yi Liang, Le Qi Chen, Tin Ho Yuen, Basak Sahin, Dragoș M. Vasilescu, Mark Trinder, Keith Walley, Patrick C.N. Rensen, John H. Boyd, Liam R. Brunham
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BACKGROUND Novel biomarkers to identify infectious patients transmitting Mycobacterium tuberculosis are urgently needed to control the global tuberculosis (TB) pandemic. We hypothesized that proteins released into the plasma in active pulmonary TB are clinically useful biomarkers to distinguish TB cases from healthy individuals and patients with other respiratory infections.METHODS We applied a highly sensitive non-depletion tandem mass spectrometry discovery approach to investigate plasma protein expression in pulmonary TB cases compared to healthy controls in South African and Peruvian cohorts. Bioinformatic analysis using linear modeling and network correlation analyses identified 118 differentially expressed proteins, significant through 3 complementary analytical pipelines. Candidate biomarkers were subsequently analyzed in 2 validation cohorts of differing ethnicity using antibody-based proximity extension assays.RESULTS TB-specific host biomarkers were confirmed. A 6-protein diagnostic panel, comprising FETUB, FCGR3B, LRG1, SELL, CD14, and ADA2, differentiated patients with pulmonary TB from healthy controls and patients with other respiratory infections with high sensitivity and specificity in both cohorts.CONCLUSION This biomarker panel exceeds the World Health Organization Target Product Profile specificity criteria for a triage test for TB. The new biomarkers have potential for further development as near-patient TB screening assays, thereby helping to close the case-detection gap that fuels the global pandemic.FUNDING Medical Research Council (MRC) (MR/R001065/1, MR/S024220/1, MR/P023754/1, and MR/W025728/1); the MRC and the UK Foreign Commonwealth and Development Office; the UK National Institute for Health Research (NIHR); the Wellcome Trust (094000, 203135, and CC2112); Starter Grant for Clinical Lecturers (Academy of Medical Sciences UK); the British Infection Association; the Program for Advanced Research Capacities for AIDS in Peru at Universidad Peruana Cayetano Heredia (D43TW00976301) from the Fogarty International Center at the US NIH; the UK Technology Strategy Board/Innovate UK (101556); the Francis Crick Institute, which receives funding from UKRI-MRC (CC2112); Cancer Research UK (CC2112); and the NIHR Biomedical Research Centre of Imperial College NHS.
Authors
Hannah F. Schiff, Naomi F. Walker, Cesar Ugarte-Gil, Marc Tebruegge, Antigoni Manousopoulou, Spiros D. Garbis, Salah Mansour, Pak Ho (Michael) Wong, Gabrielle Rockett, Paolo Piazza, Mahesan Niranjan, Andres F. Vallejo, Christopher H. Woelk, Robert J. Wilkinson, Liku B. Tezera, Diana Garay-Baquero, Paul Elkington
×Abstract
Joint injury is associated with risk for development of osteoarthritis (OA). Increasing evidence suggests that activation of fibrinolysis is involved in OA pathogenesis. However, the role of the fibrinolytic pathway is not well understood. Here, we showed that the fibrinolytic pathway, which includes plasminogen/plasmin, tissue plasminogen activator, urokinase plasminogen activator (uPA), and the uPA receptor (uPAR), was dysregulated in human OA joints. Pharmacological inhibition of plasmin attenuated OA progression after a destabilization of the medial meniscus in a mouse model whereas genetic deficiency of plasmin activator inhibitor, or injection of plasmin, exacerbated OA. We detected increased uptake of uPA/uPAR in mouse OA joints by microPET/CT imaging. In vitro studies identified that plasmin promotes OA development through multiple mechanisms, including the degradation of lubricin and cartilage proteoglycans and induction of inflammatory and degradative mediators. We showed that uPA and uPAR produced inflammatory and degradative mediators by activating the PI3K, 3′-phosphoinositide-dependent kinase-1, AKT, and ERK signaling cascades and activated matrix metalloproteinases to degrade proteoglycan. Together, we demonstrated that fibrinolysis contributes to the development of OA through multiple mechanisms and suggested that therapeutic targeting of the fibrinolysis pathway can prevent or slow development of OA.
Authors
Qian Wang, Guoqiang Shao, Xiaoyi Zhao, Heidi H. Wong, Kate Chin, Mackenzie Zhao, Audrey Bai, Michelle S. Bloom, Zelda Z. Love, Constance R. Chu, Zhen Cheng, William H. Robinson
×Abstract
Acute myeloid leukemia (AML) is a fatal disease characterized by the accumulation of undifferentiated myeloblasts, and agents that promote differentiation have been effective in this disease but are not curative. Dihydroorotate dehydrogenase inhibitors (DHODHi) have the ability to promote AML differentiation and target aberrant malignant myelopoiesis. We introduce HOSU-53, a DHODHi with significant monotherapy activity, which is further enhanced when combined with other standard-of-care therapeutics. We further discovered that DHODHi modulated surface expression of CD38 and CD47, prompting the evaluation of HOSU-53 combined with anti-CD38 and anti-CD47 therapies, where we identified a compelling curative potential in an aggressive AML model with CD47 targeting. Finally, we explored using plasma dihydroorotate (DHO) levels to monitor HOSU-53 safety and found that the level of DHO accumulation could predict HOSU-53 intolerability, suggesting the clinical use of plasma DHO to determine safe DHODHi doses. Collectively, our data support the clinical translation of HOSU-53 in AML, particularly to augment immune therapies. Potent DHODHi to date have been limited by their therapeutic index; however, we introduce pharmacodynamic monitoring to predict tolerability while preserving antitumor activity. We additionally suggest that DHODHi is effective at lower doses with select immune therapies, widening the therapeutic index.
Authors
Ola A. Elgamal, Sydney Fobare, Sandip Vibhute, Abeera Mehmood, Dennis C. Vroom, Mariah L. Johnson, Blaise Stearns, James R. Lerma, Jean Truxall, Emily Stahl, Bridget Carmichael, Shelley J. Orwick, Alice S. Mims, Emily Curran, Ramasamy Santhanam, Susheela Tridandapani, Mitch A. Phelps, Zhiliang Xie, Christopher C. Coss, Sharyn D. Baker, Jeffrey Patrick, Janel K. Ezzell, Jayesh Rai, Jianmin Pan, Shesh N. Rai, Cody Stillwell, Mark Wunderlich, Mouad Abdulrahim, Thomas E. Goodwin, Gerard Hilinski, Chad E. Bennett, Erin Hertlein, John C. Byrd
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Both anaplastic thyroid cancer (ATC) and papillary thyroid cancer (PTC) originate from thyroid follicular epithelial cells, but ATC has a significantly worse prognosis and shows resistance to conventional therapies. However, clinical trials found that immunotherapy works better in ATC than late-stage PTC. Here, we used single-cell RNA sequencing (scRNA-Seq) to generate a single-cell atlas of thyroid cancer. Differences in ATC and PTC tumor microenvironment components (including malignant cells, stromal cells, and immune cells) leading to the polarized prognoses were identified. Intriguingly, we found that CXCL13+ T lymphocytes were enriched in ATC samples and might promote the development of early tertiary lymphoid structure (TLS). Last, murine experiments and scRNA-Seq analysis of a treated patient’s tumor demonstrated that famitinib plus anti–PD-1 antibody could advance TLS in thyroid cancer. We displayed the cellular landscape of ATC and PTC, finding that CXCL13+ T cells and early TLS might make ATC more sensitive to immunotherapy.
Authors
Pei-Zhen Han, Wei-Dong Ye, Peng-Cheng Yu, Li-Cheng Tan, Xiao Shi, Xu-Feng Chen, Cong He, Jia-Qian Hu, Wen-Jun Wei, Zhong-Wu Lu, Ning Qu, Yu Wang, Qing-Hai Ji, Dong-Mei Ji, Yu-Long Wang
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Inherited retinal dystrophies (IRDs) are progressive diseases leading to vision loss. Mutation in the eyes shut homolog (EYS) gene is one of the most frequent causes of IRD. However, the mechanism of photoreceptor cell degeneration by mutant EYS has not been fully elucidated. Here, we generated retinal organoids from induced pluripotent stem cells (iPSCs) derived from patients with EYS-associated retinal dystrophy (EYS-RD). In photoreceptor cells of RD organoids, both EYS and G protein–coupled receptor kinase 7 (GRK7), one of the proteins handling phototoxicity, were not in the outer segment, where they are physiologically present. Furthermore, photoreceptor cells in RD organoids were vulnerable to light stimuli, and especially to blue light. Mislocalization of GRK7, which was also observed in eys-knockout zebrafish, was reversed by delivering control EYS into photoreceptor cells of RD organoids. These findings suggest that avoiding phototoxicity would be a potential therapeutic approach for EYS-RD.
Authors
Yuki Otsuka, Keiko Imamura, Akio Oishi, Kazuhide Asakawa, Takayuki Kondo, Risako Nakai, Mika Suga, Ikuyo Inoue, Yukako Sagara, Kayoko Tsukita, Kaori Teranaka, Yu Nishimura, Akira Watanabe, Kazuhiro Umeyama, Nanako Okushima, Kohnosuke Mitani, Hiroshi Nagashima, Koichi Kawakami, Keiko Muguruma, Akitaka Tsujikawa, Haruhisa Inoue
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HIPK2 is a multifunctional kinase that acts as a key pathogenic mediator of chronic kidney disease and fibrosis. It acts as a central effector of multiple signaling pathways implicated in kidney injury, such as TGF-β/Smad3-mediated extracellular matrix accumulation, NF-κB–mediated inflammation, and p53-mediated apoptosis. Thus, a better understanding of the specific HIPK2 regions necessary for distinct downstream pathway activation is critical for optimal drug development for CKD. Our study now shows that caspase-6–mediated removal of the C-terminal region of HIPK2 (HIPK2-CT) lead to hyperactive p65 NF-κB transcriptional response in kidney cells. In contrast, the expression of cleaved HIPK2-CT fragment could restrain the NF-κB transcriptional activity by cytoplasmic sequestration of p65 and the attenuation of IκBα degradation. Therefore, we examined whether HIPK2-CT expression can be exploited to restrain renal inflammation in vivo. The induction of HIPK2-CT overexpression in kidney tubular cells attenuated p65 nuclear translocation, expression of inflammatory cytokines, and macrophage infiltration in the kidneys of mice with unilateral ureteral obstruction and LPS-induced acute kidney injury. Collectively, our findings indicate that the HIPK2-CT is involved in the regulation of nuclear NF-κB transcriptional activity and that HIPK2-CT or its analogs could be further exploited as potential antiinflammatory agents to treat kidney disease.
Authors
Ye Feng, Zhengzhe Li, Heather Wang, Bi-Cheng Liu, Kyung Lee, John Cijiang He
×Abstract
Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2, a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1-KO cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global KO of Spns1 caused embryonic lethality between E12.5 and E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1-KO mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K/AKT signaling pathway. Furthermore, we identified 3 human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.
Authors
Hoa T.T. Ha, SiYi Liu, Xuan T.A. Nguyen, Linh K. Vo, Nancy C.P. Leong, Dat T. Nguyen, Shivaranjani Balamurugan, Pei Yen Lim, YaJun Wu, Eunju Seong, Toan Q. Nguyen, Jeongah Oh, Markus R. Wenk, Amaury Cazenave-Gassiot, Zuhal Yapici, Wei-Yi Ong, Margit Burmeister, Long N. Nguyen
×Abstract
Excessive lipolysis in white adipose tissue (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In humans, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocyte lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicated that mice lacking P2Y13-R showed a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared with their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R–knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.
Authors
Thibaut Duparc, Emilia Gore, Guillaume Combes, Diane Beuzelin, Julie Pires Da Silva, Vanessa Bouguetoch, Marie-Adeline Marquès, Ana Velazquez, Nathalie Viguerie, Geneviève Tavernier, Peter Arner, Mikael Rydén, Dominique Langin, Nabil Sioufi, Mohamad Nasser, Cendrine Cabou, Souad Najib, Laurent O. Martinez
×Abstract
Allergic airway disease (AAD) is an example of type 2 inflammation that leads to chronic airway eosinophilia controlled by CD4 Th2 cells. Inflammation is reinforced by mast cells and basophils armed with allergen-specific IgE made by allergen-specific B2 B cells of the adaptive immune system. Little is known about how AAD is affected by innate B1 cells, which produce natural antibodies (NAbs) that facilitate apoptotic cell clearance and detect damage- and pathogen-associated molecular patterns (DAMPS and PAMPS). We used transgenic mice lacking either B cells or NAbs in distinct mouse models of AAD that require either DAMPS or PAMPS as the initial trigger for type 2 immunity. In a DAMP-induced allergic model, driven by alum and uric acid, mouse strains lacking B cells (CD19DTA), NAbs (IgHEL MD4), or all secreted antibodies (sIgm–/–Aid–/–) displayed a significant reduction in both eosinophilia and Th2 priming compared with WT or Aid–/– mice lacking only germinal center–dependent high-affinity class-switched antibodies. Replenishing B cell–deficient mice with either unimmunized B1 B cells or NAbs during sensitization restored eosinophilia, suggesting that NAbs are required for licensing antigen-presenting cells to prime type 2 immunity. Conversely, PAMP-dependent type 2 priming to house dust mite or Aspergillus was not dependent on NAbs. This study reveals an underappreciated role of B1 B cell–generated NAbs in selectively driving DAMP-induced type 2 immunity.
Authors
Arlind B. Mara, Kavita Rawat, William T. King, Claudia V. Jakubzick
×Abstract
The polymerization of myosin molecules into thick filaments in muscle sarcomeres is essential for cardiac contractility, with the attenuation of interactions between the heads of myosin molecules within the filaments being proposed to result in hypercontractility, as observed in hypertrophic cardiomyopathy (HCM). However, experimental evidence demonstrates that the structure of these giant macromolecular complexes is highly dynamic, with molecules exchanging between the filaments and a pool of soluble molecules on the minute timescale. Therefore, we sought to test the hypothesis that the enhancement of interactions between the heads of myosin molecules within thick filaments limits the mobility of myosin by taking advantage of mavacamten, a small molecule approved for the treatment of HCM. Myosin molecules were labeled in vivo with a green fluorescent protein (GFP) and imaged in intact hearts using multiphoton microscopy. Treatment of the intact hearts with mavacamten resulted in an unexpected > 5-fold enhancement in GFP-myosin mobility within the sarcomere. In vitro biochemical assays suggested that mavacamten enhanced the mobility of GFP-myosin by increasing the solubility of myosin molecules, through the stabilization of a compact/folded conformation of the molecules, once disassociated from the thick filaments. These findings provide alternative insight into the mechanisms by which molecules exchange into and out of thick filaments and have implications for how mavacamten may affect cardiac contractility.
Authors
Colleen M. Kelly, Jody L. Martin, Michael J. Previs
×Abstract
BACKGROUND COVID-19 convalescent plasma (CCP) virus-specific antibody levels that translate into recipient posttransfusion antibody levels sufficient to prevent disease progression are not defined.METHODS This secondary analysis correlated donor and recipient antibody levels to hospitalization risk among unvaccinated, seronegative CCP recipients within the outpatient, double-blind, randomized clinical trial that compared CCP to control plasma. The majority of COVID-19 CCP arm hospitalizations (15/17, 88%) occurred in this unvaccinated, seronegative subgroup. A functional cutoff to delineate recipient high versus low posttransfusion antibody levels was established by 2 methods: (i) analyzing virus neutralization–equivalent anti–Spike receptor-binding domain immunoglobulin G (anti-S-RBD IgG) responses in donors or (ii) receiver operating characteristic (ROC) curve analysis.RESULTS SARS-CoV-2 anti–S-RBD IgG antibody was volume diluted 21.3-fold into posttransfusion seronegative recipients from matched donor units. Virus-specific antibody delivered was approximately 1.2 mg. The high-antibody recipients transfused early (symptom onset within 5 days) had no hospitalizations. A CCP-recipient analysis for antibody thresholds correlated to reduced hospitalizations found a statistical significant association between early transfusion and high antibodies versus all other CCP recipients (or control plasma), with antibody cutoffs established by both methods-donor-based virus neutralization cutoffs in posttransfusion recipients (0/85 [0%] versus 15/276 [5.6%]; P = 0.03) or ROC-based cutoff (0/94 [0%] versus 15/267 [5.4%]; P = 0.01).CONCLUSION In unvaccinated, seronegative CCP recipients, early transfusion of plasma units in the upper 30% of study donors’ antibody levels reduced outpatient hospitalizations. High antibody level plasma units, given early, should be reserved for therapeutic use.TRIAL REGISTRATION ClinicalTrials.gov NCT04373460.FUNDING Department of Defense (W911QY2090012); Defense Health Agency; Bloomberg Philanthropies; the State of Maryland; NIH (3R01AI152078-01S1, U24TR001609-S3, 1K23HL151826NIH); the Mental Wellness Foundation; the Moriah Fund; Octapharma; the Healthnetwork Foundation; the Shear Family Foundation; the NorthShore Research Institute; and the Rice Foundation.
Authors
Han-Sol Park, Anna Yin, Caelan Barranta, John S. Lee, Christopher A. Caputo, Jaiprasath Sachithanandham, Maggie Li, Steve Yoon, Ioannis Sitaras, Anne Jedlicka, Yolanda Eby, Malathi Ram, Reinaldo E. Fernandez, Owen R. Baker, Aarthi G. Shenoy, Giselle S. Mosnaim, Yuriko Fukuta, Bela Patel, Sonya L. Heath, Adam C. Levine, Barry R. Meisenberg, Emily S. Spivak, Shweta Anjan, Moises A. Huaman, Janis E. Blair, Judith S. Currier, James H. Paxton, Jonathan M. Gerber, Joann R. Petrini, Patrick B. Broderick, William Rausch, Marie Elena Cordisco, Jean Hammel, Benjamin Greenblatt, Valerie C. Cluzet, Daniel Cruser, Kevin Oei, Matthew Abinante, Laura L. Hammitt, Catherine G. Sutcliffe, Donald N. Forthal, Martin S. Zand, Edward R. Cachay, Jay S. Raval, Seble G. Kassaye, Christi E. Marshall, Anusha Yarava, Karen Lane, Nichol A. McBee, Amy L. Gawad, Nicky Karlen, Atika Singh, Daniel E. Ford, Douglas A. Jabs, Lawrence J. Appel, David M. Shade, Bryan Lau, Stephan Ehrhardt, Sheriza N. Baksh, Janna R. Shapiro, Jiangda Ou, Yu Bin Na, Maria D. Knoll, Elysse Ornelas-Gatdula, Netzahualcoyotl Arroyo-Curras, Thomas J. Gniadek, Patrizio Caturegli, Jinke Wu, Nelson Ndahiro, Michael J. Betenbaugh, Alyssa Ziman, Daniel F. Hanley, Arturo Casadevall, Shmuel Shoham, Evan M. Bloch, Kelly A. Gebo, Aaron A.R. Tobian, Oliver Laeyendecker, Andrew Pekosz, Sabra L. Klein, David J. Sullivan
×Abstract
BACKGROUND Survivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODS We measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTS Microglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19–specific cytokines.CONCLUSION Prolonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDING SCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.
Authors
Rogan A. Grant, Taylor A. Poor, Lango Sichizya, Estefani Diaz, Joseph I. Bailey, Sahil Soni, Karolina J. Senkow, Xóchitl G. Pérez-Leonor, Hiam Abdala-Valencia, Ziyan Lu, Helen K. Donnelly, Lacy M. Simons, Egon A. Ozer, Robert M. Tighe, Jon W. Lomasney, Richard G. Wunderink, Benjamin D. Singer, Alexander V. Misharin, G.R. Scott Budinger, for The Northwestern University Successful Clinical Response In Pneumonia Therapy (NU SCRIPT) Investigators
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Abstract
Dysregulated lipid homeostasis is emerging as a potential cause of neurodegenerative disorders. However, evidence of errors in lipid homeostasis as a pathogenic mechanism of neurodegeneration remains limited. Here, we show that cerebellar neurodegeneration caused by Sorting Nexin 14 (SNX14) deficiency is associated with lipid homeostasis defects. Recent studies indicate that SNX14 is an inter-organelle lipid transfer protein that regulates lipid transport, lipid droplet (LD) biogenesis, and fatty acid desaturation, suggesting that human SNX14 deficiency belongs to an expanding class of cerebellar neurodegenerative disorders caused by altered cellular lipid homeostasis. To test this hypothesis, we generated a mouse model that recapitulates human SNX14 deficiency at a genetic and phenotypic level. We demonstrate that cerebellar Purkinje cells (PCs) are selectively vulnerable to SNX14 deficiency while forebrain regions preserve their neuronal content. Ultrastructure and lipidomic studies reveal widespread lipid storage and metabolism defects in SNX14 deficient mice. However, pre-degenerating SNX14 deficient cerebella show a unique accumulation of acylcarnitines and depletion of triglycerides. Furthermore, defects in LD content and telolysosome enlargement in pre-degenerating PCs, suggest lipotoxicity as a pathogenic mechanism of SNX14 deficiency. Our work shows a selective cerebellar vulnerability to altered lipid homeostasis and provides a mouse model for future therapeutic studies.
Authors
Yijing Zhou, Vanessa B. Sanchez, Peining Xu, Thomas Roule, Marco Flores-Mendez, Brianna Ciesielski, Donna Yoo, Hiab Teshome, Teresa Jimenez, Shibo Liu, Mike Henne, Tim O’Brien, Ye He, Clementina Mesaros, Naiara Akizu
Abstract
Background. Upper body obesity (UBO) results in insulin resistance with regards to free fatty acid (FFA) release; how this differs by fat depot and sex between UBO and lean adults is unknown. We tested the hypothesis that insulin suppression of FFA release from the splanchnic bed, leg fat and upper body non-splanchnic (UBNS) adipose tissue would be impaired in UBO. Methods. Fourteen UBO (7 men, 7 women) and 14 healthy, normal weight (7 men, 7 women) volunteers participated in studies that included femoral artery, femoral vein and hepatic vein catheterization. We then measured leg and splanchnic plasma flow as well as FFA kinetics (using isotopic tracers) under overnight fasting, low- and high-dose insulin infusion using the insulin clamp technique. Results. We found the expected insulin resistance in UBO; the most quantitatively important difference between UBO and lean adults was greater FFA release from UBNS adipose tissue when plasma insulin concentrations are in the post-prandial, physiological range. There were obesity, but not sex differences in the regulation of splanchnic FFA release and sex differences in the regulation of leg FFA release. Conclusion. Reversing the defects in insulin-regulated UBNS adipose tissue FFA release would have the greatest impact on systemic FFA abnormalities in UBO. Trial Registration: (not applicable) Funding: These studies were supported by grants DK45343 and DK40484 from the U.S. Public Health Service, and the Novo Nordic Foundation (grant numbers NNF18OC0031804 and NNF16OC0021406) and the Independent Research Fund Denmark (grant number 8020-00420B).
Authors
Søren Nielsen, Michael D. Jensen
Abstract
Allogeneic hematopoietic stem cell transplantation (aHSCT) can cure patients with otherwise fatal leukemias and lymphomas. However, the benefits of aHSCT are limited by graft-versus-host disease (GVHD). Minnelide, a water-soluble analog of triptolide, has demonstrated potent anti-inflammatory and anti-tumor activity in several pre-clinical models and has proven both safe and efficacious in clinical trials for advanced gastro-intestinal malignancies. Here, we tested the effectiveness of Minnelide in preventing acute GVHD as compared to cyclophosphamide post-aHSCT (PTCy). Strikingly, we found Minnelide improved survival, weight loss and clinical scores in an MHC-mismatched model of aHSCT. These benefits were also apparent in minor MHC-matched aHSCT and xenogeneic HSCT models. Minnelide was comparable to PTCy in terms of survival, GVHD clinical score and colonic length. Notably, in addition to decreased donor T cell infiltration early post-HSCT, several regulatory cell populations including Tregs, ILC2s and MDSCs in the colon were increased which together may account for Minnelide’s GVHD suppression post-HSCT. Importantly, Minnelide GVHD prevention was accompanied by preservation of graft-versus-tumor (GVT) activity. As Minnelide possesses anti-AML activity and is being applied in clinical trials, together with the present findings, we conclude that this compound might provide a new approach for AML patients undergoing aHSCT.
Authors
Sabrina N. Copsel, Vanessa T. Garrido, Henry Barreras, Cameron S. Bader, Brent Pfeiffer, Beatriz Mateo-Victoriano, Dietlinde Wolf, Miguel Gallardo, Sophie Paczesny, Krishna V. Komanduri, Cara L. Benjamin, Alejandro Villarino, Ashok K. Saluja, Robert B. Levy
Intimomedial tears of the aorta heal by smooth muscle cell-mediated fibrosis without atherosclerosis
Abstract
Background. Disease of the aorta varies from atherosclerosis to aneurysms with complications including rupture, dissection, and poorly characterized limited tears. We studied limited tears without any mural hematoma, termed intimomedial tears to gain insight into aortic vulnerability to excessive wall stresses. Our premise is that minimal injuries in aortas with sufficient medial resilience to prevent tear progression correspond to initial mechanisms leading to complete structural failure in aortas with significantly compromised medial resilience. Methods. Intimomedial tears were macroscopically identified in 9 of 108 ascending aortas after surgery and analyzed by histology and immunofluorescence confocal microscopy. Results. Non-hemorrhagic, non atheromatous tears correlated with advanced aneurysmal disease and most lacked distinctive symptoms or radiological signs. Tears traversed the intima and part of the subjacent media, while the resultant defects were partially or completely filled with neointima characterized by differentiated smooth muscle cells, scattered leukocytes, dense fibrosis, and absent elastic laminae despite tropoelastin synthesis. Healed lesions contained organized fibrin at tear edges without evidence of plasma and erythrocyte extravasation or lipid accumulation. Conclusion. These findings suggest a multiphasic model of aortic wall failure in which primary lesions of intimomedial tears either heal if the media is sufficiently resilient or progress as dissection or rupture by medial delamination and tear completion, respectively. Moreover, mural incorporation of thrombus and cellular responses to injury, two historically important concepts in atheroma pathogenesis, contribute to vessel wall repair with adequate conduit function but even together are not sufficient to induce atherosclerosis. Funding. R01-HL146723, R01-HL168473, and Yale Department of Surgery.
Authors
Abdulrahman H.M. Hassab, David J. Hur, Prashanth Vallabhajosyula, George Tellides, Roland Assi
Abstract
Recent studies have uncovered that non-coding sequence variants may relate to Axenfeld-Rieger syndrome (ARS), a rare developmental anomaly with genetic heterogeneity. However, how these genomic regions are functionally and structurally associated with ARS is still unclear. In this study, we performed genome-wide linkage analysis and whole-genome sequencing in a Chinese ARS family and identified a heterozygous deletion of about 570 kb (termed LOH-1) in the intergenic sequence between PITX2 and FAM241A. Knockout of LOH-1 homologous sequences caused ARS phenotypes in mice. RNA-seq and RT-qPCR revealed a significant reduction in Pitx2 gene expression in LOH-1–/– mice, while Foxc1 expression remained unchanged. ChIP-seq and bioinformatics analysis identified a potential enhancer region (LOH-E1) within LOH-1. Deletion of LOH-E1 led to a significant downregulation of the PITX2 gene. Mechanistically, we found a sequence (hg38 chr4:111,399,594-111,399,691) which is on LOH-E1 could regulate PITX2 by binding to RAD21, a critical component of the cohesin complex. Knockdown of RAD21 resulted in reduced PITX2 expression. Collectively, our findings indicate that a potential enhancer sequence which is within LOH-1 may regulate PITX2 expression remotely through cohesin-mediated loop domains, leading to ARS when absent. 2
Authors
Yizheng Jiang, Yu Peng, Qi Tian, Zhe Cheng, Bei Feng, Junping Hu, Lu Xia, Hui Guo, Kun Xia, Liang Zhou, Zhengmao Hu
