Issue published February 22, 2024

  • On the cover: Inhibiting centrosome clustering reduces cystogenesis and improves kidney function in autosomal dominant polycystic kidney disease
  • Cheng et al. demonstrate that blocking centrosome clustering in kidney epithelia that contain excess centrosomes stops their proliferation, which results in improved renal morphology and function in autosomal dominant polycystic kidney disease mouse models and patient-derived cells. The cover image shows immunofluorescence staining of a developing embryonic mouse kidney. Image credit: Ewa Langner.

Research Articles
Abstract

Different from the well-studied canonical NF-κB member RelA, the role of the noncanonical NF-κB member NF-κB2 in solid tumors, and lung cancer in particular, is poorly understood. Here we report that in contrast to the tumor-promoting role of RelA, NF-κB2 intrinsic to lung epithelial and tumor cells had no marked effect on lung tumorigenesis and progression. On the other hand, NF-κB2 limited dendritic cell number and activation in the lung but protected lung macrophages and drove them to promote lung cancer through controlling activation of noncanonical and canonical NF-κB, respectively. NF-κB2 was also required for B cell maintenance and T cell activation. The antitumor activity of lymphocyte NF-κB2 was dominated by the protumor function of myeloid NF-κB2; thus, NF-κB2 has an overall tumor-promoting activity. These studies reveal a cell type–dependent role for NF-κB2 in lung cancer and help understand the complexity of NF-κB action and lung cancer pathogenesis for better design of NF-κB–targeted therapy against this deadliest cancer.

Authors

Fan Sun, Yadong Xiao, Steven D. Shapiro, Zhaoxia Qu, Gutian Xiao

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Abstract

The lipidome of immune cells during infection has remained unexplored, although evidence of the importance of lipids in the context of immunity is mounting. In this study, we performed untargeted lipidomic analysis of blood monocytes and neutrophils from patients hospitalized for pneumonia and age- and sex-matched noninfectious control volunteers. We annotated 521 and 706 lipids in monocytes and neutrophils, respectively, which were normalized to an extensive set of internal standards per lipid class. The cellular lipidomes were profoundly altered in patients, with both common and distinct changes between the cell types. Changes involved every level of the cellular lipidome: differential lipid species, class-wide shifts, and altered saturation patterns. Overall, differential lipids were mainly less abundant in monocytes and more abundant in neutrophils from patients. One month after hospital admission, lipidomic changes were fully resolved in monocytes and partially in neutrophils. Integration of lipidomic and concurrently collected transcriptomic data highlighted altered sphingolipid metabolism in both cell types. Inhibition of ceramide and sphingosine-1-phosphate synthesis in healthy monocytes and neutrophils resulted in blunted cytokine responses upon stimulation with lipopolysaccharide. These data reveal major lipidomic remodeling in immune cells during infection, and link the cellular lipidome to immune functionality.

Authors

Alex R. Schuurman, Osoul Chouchane, Joe M. Butler, Hessel Peters-Sengers, Sebastiaan Joosten, Xanthe Brands, Bastiaan W. Haak, Natasja A. Otto, Fabrice Uhel, Augustijn Klarenbeek, Christine C.A. van Linge, Antoine van Kampen, Mia Pras-Raves, Michel van Weeghel, Marco van Eijk, Maria J. Ferraz, Daniël R. Faber, Alex de Vos, Brendon P. Scicluna, Frédéric M. Vaz, W. Joost Wiersinga, Tom van der Poll

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Abstract

Functional avidity is supposed to critically shape the quality of immune responses, thereby influencing host protection against infectious agents including SARS-CoV-2. Here we show that after human SARS-CoV-2 vaccination, a large portion of high-avidity spike-specific CD4+ T cells lost CD3 expression after in vitro activation. The CD3– subset was enriched for cytokine-positive cells, including elevated per-cell expression levels, and showed increased polyfunctionality. Assessment of key metabolic pathways by flow cytometry revealed that superior functionality was accompanied by a shift toward fatty acid synthesis at the expense of their oxidation, whereas glucose transport and glycolysis were similarly regulated in SARS-CoV-2–specific CD3– and CD3+ subsets. As opposed to their CD3+ counterparts, frequencies of vaccine-specific CD3– T cells positively correlated with both the size of the naive CD4+ T cell pool and vaccine-specific IgG levels. Moreover, their frequencies negatively correlated with advancing age and were impaired in patients under immunosuppressive therapy. Typical recall antigen–reactive T cells showed a comparable segregation into functionally and metabolically distinct CD3+ and CD3– subsets but were quantitatively maintained upon aging, likely due to earlier recruitment in life. In summary, our data identify CD3– T helper cells as correlates of high-quality immune responses that are impaired in at-risk populations.

Authors

Arne Sattler, Stefanie Gamradt, Vanessa Proß, Linda Marie Laura Thole, An He, Eva Vanessa Schrezenmeier, Katharina Jechow, Stefan M. Gold, Sören Lukassen, Christian Conrad, Katja Kotsch

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Abstract

The clinical spectrum of thyrotropin receptor–mediated (TSHR-mediated) diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with 1 CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in 2 families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants as well as distinct common noncoding TSHR SNPs significantly associated with thyroid phenotypes, but there was no other significant association between TSHR variants and more than 2,000 nonthyroid disease endpoints. Finally, our TSHR M453T–knockin model revealed that the phenotype was dependent on the mutation’s signaling properties and was ameliorated by increased iodine intake. In summary, our data show that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region as well as by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.

Authors

Kristiina Makkonen, Meeri Jännäri, Luís Crisóstomo, Matilda Kuusi, Konrad Patyra, Vladyslav Melnyk, Veli Linnossuo, Johanna Ojala, Rowmika Ravi, Christoffer Löf, Juho-Antti Mäkelä, Päivi Miettinen, Saila Laakso, Marja Ojaniemi, Jarmo Jääskeläinen, Markku Laakso, Filip Bossowski, Beata Sawicka, Karolina Stożek, Artur Bossowski, Gunnar Kleinau, Patrick Scheerer, FinnGen FinnGen, Mary Pat Reeve, Jukka Kero

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Abstract

A robust, sterile inflammation underlies myocardial ischemia and reperfusion injury (MIRI). Several subsets of B cells possess the immunoregulatory capacity that limits tissue damage, yet the role of B cells in MIRI remains elusive. Here, we sought to elucidate the contribution of B cells to MIRI by transient ligation of the left anterior descending coronary artery in B cell–depleted or –deficient mice. Following ischemia and reperfusion (I/R), regulatory B cells are rapidly recruited to the heart. B cell–depleted or –deficient mice exhibited exacerbated tissue damage, adverse cardiac remodeling, and an augmented inflammatory response after I/R. Rescue and chimeric experiments indicated that the cardioprotective effect of B cells was not solely dependent on IL-10. Coculture experiments demonstrated that B cells induced neutrophil apoptosis through contact-dependent interactions, subsequently promoting reparative macrophage polarization by facilitating the phagocytosis of neutrophils by macrophages. The in vivo cardioprotective effect of B cells was undetectable in the absence of neutrophils after I/R. Mechanistically, ligand-receptor imputation identified FCER2A as a potential mediator of interactions between B cells and neutrophils. Blocking FCER2A on B cells resulted in a reduction in the percentage of apoptotic neutrophils, contributing to the deterioration of cardiac remodeling. Our findings unveil a potential cardioprotective role of B cells in MIRI through mechanisms involving FCER2A, neutrophils, and macrophages.

Authors

Fangyang Huang, Jialiang Zhang, Hao Zhou, Tianyi Qu, Yan Wang, Kexin Jiang, Yutong Liu, Yuanning Xu, Mao Chen, Li Chen

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Abstract

Crohn’s disease (CD) is a chronic inflammatory gut disorder. Molecular mechanisms underlying the clinical heterogeneity of CD remain poorly understood. MicroRNAs (miRNAs) are important regulators of gut physiology, and several have been implicated in the pathogenesis of adult CD. However, there is a dearth of large-scale miRNA studies for pediatric CD. We hypothesized that specific miRNAs uniquely mark pediatric CD. We performed small RNA-Seq of patient-matched colon and ileum biopsies from treatment-naive pediatric patients with CD (n = 169) and a control cohort (n = 108). Comprehensive miRNA analysis revealed 58 miRNAs altered in pediatric CD. Notably, multinomial logistic regression analysis revealed that index levels of ileal miR-29 are strongly predictive of severe inflammation and stricturing. Transcriptomic analyses of transgenic mice overexpressing miR-29 show a significant reduction of the tight junction protein gene Pmp22 and classic Paneth cell markers. The dramatic loss of Paneth cells was confirmed by histologic assays. Moreover, we found that pediatric patients with CD with elevated miR-29 exhibit significantly lower Paneth cell counts, increased inflammation scores, and reduced levels of PMP22. These findings strongly indicate that miR-29 upregulation is a distinguishing feature of pediatric CD, highly predictive of severe phenotypes, and associated with inflammation and Paneth cell loss.

Authors

Alexandria J. Shumway, Michael T. Shanahan, Emilie Hollville, Kevin Chen, Caroline Beasley, Jonathan W. Villanueva, Sara Albert, Grace Lian, Moises R. Cure, Matthew Schaner, Lee-Ching Zhu, Surekha Bantumilli, Mohanish Deshmukh, Terrence S. Furey, Shehzad Z. Sheikh, Praveen Sethupathy

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Abstract

Circular RNAs (circRNAs) are highly expressed in the mammalian intestinal epithelium, but their functions remain largely unknown. Here, we identified the circRNA Cdr1as as a repressor of intestinal epithelial regeneration and defense. Cdr1as levels increased in mouse intestinal mucosa after colitis and septic stress, as well as in human intestinal mucosa from patients with inflammatory bowel disease and sepsis. Ablation of the Cdr1as locus from the mouse genome enhanced renewal of the intestinal mucosa, promoted injury-induced epithelial regeneration, and protected the mucosa against colitis. We found approximately 40 microRNAs, including miR-195, differentially expressed between intestinal mucosa of Cdr1as-knockout (Cdr1as–/–) versus littermate mice. Increasing the levels of Cdr1as inhibited intestinal epithelial repair after wounding in cultured cells and repressed growth of intestinal organoids cultured ex vivo, but this inhibition was abolished by miR-195 silencing. The reduction in miR-195 levels in the Cdr1as–/– intestinal epithelium was the result of reduced stability and processing of the precursor miR-195. These findings indicate that Cdr1as reduces proliferation and repair of the intestinal epithelium at least in part via interaction with miR-195 and highlight a role for induced Cdr1as in the pathogenesis of unhealed wounds and disrupted renewal of the intestinal mucosa.

Authors

Hee Kyoung Chung, Lan Xiao, Naomi Han, Jason Chen, Vivian Yao, Cassandra M. Cairns, Benjamin Raufman, Jaladanki N. Rao, Douglas J. Turner, Rosemary Kozar, Myriam Gorospe, Jian-Ying Wang

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Abstract

Hypercapnia, elevation of the partial pressure of CO2 in blood and tissues, is a risk factor for mortality in patients with severe acute and chronic lung diseases. We previously showed that hypercapnia inhibits multiple macrophage and neutrophil antimicrobial functions and that elevated CO2 increases the mortality of bacterial and viral pneumonia in mice. Here, we show that normoxic hypercapnia downregulates innate immune and antiviral gene programs in alveolar macrophages (AMØs). We also show that zinc finger homeobox 3 (Zfhx3) — a mammalian ortholog of zfh2, which mediates hypercapnic immune suppression in Drosophila — is expressed in mouse and human macrophages. Deletion of Zfhx3 in the myeloid lineage blocked the suppressive effect of hypercapnia on immune gene expression in AMØs and decreased viral replication, inflammatory lung injury, and mortality in hypercapnic mice infected with influenza A virus. To our knowledge, our results establish Zfhx3 as the first known mammalian mediator of CO2 effects on immune gene expression and lay the basis for future studies to identify therapeutic targets to interrupt hypercapnic immunosuppression in patients with advanced lung disease.

Authors

S. Marina Casalino-Matsuda, Fei Chen, Francisco J. Gonzalez-Gonzalez, Hiroaki Matsuda, Aisha Nair, Hiam Abdala-Valencia, G.R. Scott Budinger, Jin-Tang Dong, Greg J. Beitel, Peter H.S. Sporn

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Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure, yet therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via “centrosome clustering,” a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with 2 inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, decreased interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

Authors

Tao Cheng, Aruljothi Mariappan, Ewa Langner, Kyuhwan Shim, Jay Gopalakrishnan, Moe R. Mahjoub

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Abstract

RNA-binding proteins (RBPs) interact with RNA and ubiquitously regulate RNA transcripts during their life cycle, playing a fundamental role in the progression of angiogenesis-related diseases. In the skeletal system, endothelium-dependent angiogenesis is indispensable for bone formation. However, the role of RBPs in endothelium-dependent bone formation is unclear. Here, we show that RBP–Y-box-binding protein 1 (YBX1) was strongly reduced in the bone vasculature of ovariectomy (OVX) mice. Endothelial cell–specific deletion of Ybx1 impaired CD31-high, endomucin-high (CD31hiEMCNhi) endothelium morphology and resulted in low bone mass whereas Ybx1 overexpression promoted angiogenesis-dependent osteogenesis and ameliorated bone loss. Mechanistically, YBX1 deletion disrupted CD31, EMCN, and bone morphogenetic protein 4 (BMP4) stability in an m5C-dependent manner and blocked endothelium-derived BMP4 release, thereby inhibiting osteogenic differentiation of bone mesenchymal stromal cells. Administration of recombinant BMP4 protein restored impaired bone formation in Ybx1 deletion mice. Tail vein injection of CD31-modified polyethylene glycol–poly (lactic-co-glycolic acid) carrying sciadopitysin, a natural YBX1 agonist, pharmacologically partially reversed CD31hiEMCNhi vessels’ decline and improved bone mass in both OVX and aging animals. These findings demonstrated the role of RBP-YBX1 in angiogenesis-dependent bone formation and provided a therapeutic approach for ameliorating osteoporosis.

Authors

Yu-Jue Li, Qi Guo, Ming-Sheng Ye, GuangPing Cai, Wen-Feng Xiao, Sheng Deng, Ye Xiao

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Abstract

The deposition of antipodocyte autoantibodies in the glomerular subepithelial space induces primary membranous nephropathy (MN), the leading cause of nephrotic syndrome worldwide. Taking advantage of the glomerulus-on-a-chip system, we modeled human primary MN induced by anti-PLA2R antibodies. Here we show that exposure of primary human podocytes expressing PLA2R to MN serum results in IgG deposition and complement activation on their surface, leading to loss of the chip permselectivity to albumin. C3a receptor (C3aR) antagonists as well as C3AR gene silencing in podocytes reduced oxidative stress induced by MN serum and prevented albumin leakage. In contrast, inhibition of the formation of the membrane-attack-complex (MAC), previously thought to play a major role in MN pathogenesis, did not affect permselectivity to albumin. In addition, treatment with a C3aR antagonist effectively prevented proteinuria in a mouse model of MN, substantiating the chip findings. In conclusion, using a combination of pathophysiologically relevant in vitro and in vivo models, we established that C3a/C3aR signaling plays a critical role in complement-mediated MN pathogenesis, indicating an alternative therapeutic target for MN.

Authors

Qi Zhang, Sofia Bin, Kelly Budge, Astgik Petrosyan, Valentina Villani, Paola Aguiari, Coralien Vink, Jack Wetzels, Hasmik Soloyan, Gaetano La Manna, Manuel Alfredo Podestà, Paolo Molinari, Sargis Sedrakyan, Kevin V. Lemley, Roger E. De Filippo, Laura Perin, Paolo Cravedi, Stefano Da Sacco

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Abstract

Dynamic regulation of cellular metabolism is important for maintaining homeostasis and can directly influence immune cell function and differentiation, including NK cell responses. Persistent HIV-1 infection leads to a state of chronic immune activation, NK cell subset redistribution, and progressive NK cell dysregulation. In this study, we examined the metabolic processes that characterize NK cell subsets in HIV-1 infection, including adaptive NK cell subpopulations expressing the activating receptor NKG2C, which expand during chronic infection. These adaptive NK cells exhibit an enhanced metabolic profile in HIV-1– individuals infected with human cytomegalovirus (HCMV). However, the bioenergetic advantage of adaptive CD57+NKG2C+ NK cells is diminished during chronic HIV-1 infection, where NK cells uniformly display reduced oxidative phosphorylation (OXPHOS). Defective OXPHOS was accompanied by increased mitochondrial depolarization, structural alterations, and increased DRP-1 levels promoting fission, suggesting that mitochondrial defects are restricting the metabolic plasticity of NK cell subsets in HIV-1 infection. The metabolic requirement for the NK cell response to receptor stimulation was alleviated upon IL-15 pretreatment, which enhanced mammalian target of rapamycin complex 1 (mTORC1) activity. IL-15 priming enhanced NK cell functionality to anti-CD16 stimulation in HIV-1 infection, representing an effective strategy for pharmacologically boosting NK cell responses.

Authors

Elia Moreno-Cubero, Aljawharah Alrubayyi, Stefan Balint, Ane Ogbe, Upkar S. Gill, Rebecca Matthews, Sabine Kinloch, Fiona Burns, Sarah L. Rowland-Jones, Persephone Borrow, Anna Schurich, Michael Dustin, Dimitra Peppa

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Abstract

BACKGROUND T cell responses are impaired in Staphylococcus aureus–infected children, highlighting a potential mechanism of immune evasion. This study tested the hypotheses that toxin-specific antibodies protect immune cells from bacterial killing and are associated with improved T cell function following infection.METHODS S. aureus–infected and healthy children (N = 33 each) were prospectively enrolled. During acute infection and convalescence, we quantified toxin-specific IgG levels by ELISA, antibody function using a cell killing assay, and functional T cell responses by ELISPOT.RESULTS There were no differences in toxin-specific IgG levels or ability to neutralize toxin-mediated immune cell killing between healthy and acutely infected children, but antibody levels and function increased following infection. Similarly, T cell function, which was impaired during acute infection, improved following infection. However, the response to infection was highly variable; up to half of children did not have improved antibody or T cell function. Serum from children with higher α-hemolysin–specific IgG levels more strongly protected immune cells against toxin-mediated killing. Importantly, children whose serum more strongly protected against toxin-mediated killing also had stronger immune responses to infection, characterized by more elicited antibodies and greater improvement in T cell function following infection.CONCLUSION This study demonstrates that, despite T cell impairment during acute infection, S. aureus elicits toxin-neutralizing antibodies. Individual antibody responses and T cell recovery are variable. These findings also suggest that toxin-neutralizing antibodies protect antigen-presenting cells and T cells, thereby promoting immune recovery. Finally, failure to elicit toxin-neutralizing antibodies may identify children at risk for prolonged T cell suppression.FUNDING NIH National Institute of Allergy and Infectious Diseases R01AI125489 and Nationwide Children’s Hospital.

Authors

Maureen Kleinhenz, Zhaotao Li, Usha Chidella, Walissa Picard, Amber Wolfe, Jill Popelka, Robin Alexander, Christopher P. Montgomery

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Abstract

AMP-activated protein kinase (AMPK) plays a crucial role in maintaining ATP homeostasis in photoreceptor neurons. AMPK is a heterotrimeric protein consisting of α, β, and γ subunits. The independent functions of the 2 isoforms of the catalytic α subunit, PRKAA1 and PRKAA2, are uncharacterized in specialized neurons, such as photoreceptors. Here, we demonstrate in mice that rod photoreceptors lacking PRKAA2, but not PRKAA1, showed altered levels of cGMP, GTP, and ATP, suggesting isoform-specific regulation of photoreceptor metabolism. Furthermore, PRKAA2-deficient mice displayed visual functional deficits on electroretinography and photoreceptor outer segment structural abnormalities on transmission electron microscopy consistent with neuronal dysfunction, but not neurodegeneration. Phosphoproteomics identified inosine monophosphate dehydrogenase (IMPDH) as a molecular driver of PRKAA2-specific photoreceptor dysfunction, and inhibition of IMPDH improved visual function in Prkaa2 rod photoreceptor–knockout mice. These findings highlight a therapeutically targetable PRKAA2 isoform–specific function of AMPK in regulating photoreceptor metabolism and function through a potentially previously uncharacterized mechanism affecting IMPDH activity.

Authors

Tae Jun Lee, Yo Sasaki, Philip A. Ruzycki, Norimitsu Ban, Joseph B. Lin, Hung-Ting Wu, Andrea Santeford, Rajendra S. Apte

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Abstract

A distinct adipose tissue distribution pattern was observed in patients with methylmalonyl-CoA mutase deficiency, an inborn error of branched-chain amino acid (BCAA) metabolism, characterized by centripetal obesity with proximal upper and lower extremity fat deposition and paucity of visceral fat, that resembles familial multiple lipomatosis syndrome. To explore brown and white fat physiology in methylmalonic acidemia (MMA), body composition, adipokines, and inflammatory markers were assessed in 46 patients with MMA and 99 matched controls. Fibroblast growth factor 21 levels were associated with acyl-CoA accretion, aberrant methylmalonylation in adipose tissue, and an attenuated inflammatory cytokine profile. In parallel, brown and white fat were examined in a liver-specific transgenic MMA mouse model (Mmut–/– TgINS-Alb-Mmut). The MMA mice exhibited abnormal nonshivering thermogenesis with whitened brown fat and had an ineffective transcriptional response to cold stress. Treatment of the MMA mice with bezafibrates led to clinical improvement with beiging of subcutaneous fat depots, which resembled the distribution seen in the patients. These studies defined what we believe to be a novel lipodystrophy phenotype in patients with defects in the terminal steps of BCAA oxidation and demonstrated that beiging of subcutaneous adipose tissue in MMA could readily be induced with small molecules.

Authors

Irini Manoli, Justin R. Sysol, PamelaSara E. Head, Madeline W. Epping, Oksana Gavrilova, Melissa K. Crocker, Jennifer L. Sloan, Stefanos A. Koutsoukos, Cindy Wang, Yiouli P. Ktena, Sophia Mendelson, Alexandra R. Pass, Patricia M. Zerfas, Victoria Hoffmann, Hilary J. Vernon, Laura A. Fletcher, James C. Reynolds, Maria G. Tsokos, Constantine A. Stratakis, Stephan D. Voss, Kong Y. Chen, Rebecca J. Brown, Ada Hamosh, Gerard T. Berry, Xiaoyuan Shawn Chen, Jack A. Yanovski, Charles P. Venditti

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Abstract

BACKGROUND While the benefits of statin therapy on atherosclerotic cardiovascular disease are clear, patients often experience mild to moderate skeletal myopathic symptoms, the mechanism for which is unknown. This study investigated the potential effect of high-dose atorvastatin therapy on skeletal muscle mitochondrial function and whole-body aerobic capacity in humans.METHODS Eight overweight (BMI, 31.9 ± 2.0) but otherwise healthy sedentary adults (4 females, 4 males) were studied before (day 0) and 14, 28, and 56 days after initiating atorvastatin (80 mg/d) therapy.RESULTS Maximal ADP-stimulated respiration, measured in permeabilized fiber bundles from muscle biopsies taken at each time point, declined gradually over the course of atorvastatin treatment, resulting in > 30% loss of skeletal muscle mitochondrial oxidative phosphorylation capacity by day 56. Indices of in vivo muscle oxidative capacity (via near-infrared spectroscopy) decreased by 23% to 45%. In whole muscle homogenates from day 0 biopsies, atorvastatin inhibited complex III activity at midmicromolar concentrations, whereas complex IV activity was inhibited at low nanomolar concentrations.CONCLUSION These findings demonstrate that high-dose atorvastatin treatment elicits a striking progressive decline in skeletal muscle mitochondrial respiratory capacity, highlighting the need for longer-term dose-response studies in different patient populations to thoroughly define the effect of statin therapy on skeletal muscle health.FUNDING NIH R01 AR071263.

Authors

Terence E. Ryan, Maria J. Torres, Chien-Te Lin, Angela H. Clark, Patricia M. Brophy, Cheryl A. Smith, Cody D. Smith, E. Matthew Morris, John P. Thyfault, P. Darrell Neufer

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Abstract

Patients with diabetes have a high risk of developing skeletal diseases accompanied by diabetic peripheral neuropathy (DPN). In this study, we isolated the role of DPN in skeletal disease with global and conditional knockout models of sterile-α and TIR-motif-containing protein-1 (Sarm1). SARM1, an NADase highly expressed in the nervous system, regulates axon degeneration upon a range of insults, including DPN. Global knockout of Sarm1 prevented DPN, but not skeletal disease, in male mice with type 1 diabetes (T1D). Female wild-type mice also developed diabetic bone disease but without DPN. Unexpectedly, global Sarm1 knockout completely protected female mice from T1D-associated bone suppression and skeletal fragility despite comparable muscle atrophy and hyperglycemia. Global Sarm1 knockout rescued bone health through sustained osteoblast function with abrogation of local oxidative stress responses. This was independent of the neural actions of SARM1, as beneficial effects on bone were lost with neural conditional Sarm1 knockout. This study demonstrates that the onset of skeletal disease occurs rapidly in both male and female mice with T1D completely independently of DPN. In addition, this reveals that clinical SARM1 inhibitors, currently being developed for treatment of neuropathy, may also have benefits for diabetic bone through actions outside of the nervous system.

Authors

Jennifer M. Brazill, Ivana R. Shen, Clarissa S. Craft, Kristann L. Magee, Jay S. Park, Madelyn Lorenz, Amy Strickland, Natalie K. Wee, Xiao Zhang, Alec T. Beeve, Gretchen A. Meyer, Jeffrey Milbrandt, Aaron DiAntonio, Erica L. Scheller

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Abstract

Genetic modifications leading to pain insensitivity phenotypes, while rare, provide invaluable insights into the molecular biology of pain and reveal targets for analgesic drugs. Pain insensitivity typically results from Mendelian loss-of-function mutations in genes expressed in nociceptive (pain-sensing) dorsal root ganglion (DRG) neurons that connect the body to the spinal cord. We document a pain insensitivity mechanism arising from gene overexpression in individuals with the rare 7q11.23 duplication syndrome (Dup7), who have 3 copies of the approximately 1.5-megabase Williams syndrome (WS) critical region. Based on parental accounts and pain ratings, people with Dup7, mainly children in this study, are pain insensitive following serious injury to skin, bones, teeth, or viscera. In contrast, diploid siblings (2 copies of the WS critical region) and individuals with WS (1 copy) show standard reactions to painful events. A converging series of human assessments and cross-species cell biological and transcriptomic studies identified 1 likely candidate in the WS critical region, STX1A, as underlying the pain insensitivity phenotype. STX1A codes for the synaptic vesicle fusion protein syntaxin1A. Excess syntaxin1A was demonstrated to compromise neuropeptide exocytosis from nociceptive DRG neurons. Taken together, these data indicate a mechanism for producing “genetic analgesia” in Dup7 and offer previously untargeted routes to pain control.

Authors

Michael J. Iadarola, Matthew R. Sapio, Amelia J. Loydpierson, Carolyn B. Mervis, Jill C. Fehrenbacher, Michael R. Vasko, Dragan Maric, Daniel P. Eisenberg, Tiffany A. Nash, J. Shane Kippenhan, Madeline H. Garvey, Andrew J. Mannes, Michael D. Gregory, Karen F. Berman

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Abstract

Infection with chikungunya virus (CHIKV) causes disruption of draining lymph node (dLN) organization, including paracortical relocalization of B cells, loss of the B cell–T cell border, and lymphocyte depletion that is associated with infiltration of the LN with inflammatory myeloid cells. Here, we found that, during the first 24 hours of infection, CHIKV RNA accumulated in MARCO-expressing lymphatic endothelial cells (LECs) in both the floor and medullary LN sinuses. The accumulation of viral RNA in the LN was associated with a switch to an antiviral and inflammatory gene expression program across LN stromal cells, and this inflammatory response — including recruitment of myeloid cells to the LN — was accelerated by CHIKV-MARCO interactions. As CHIKV infection progressed, both floor and medullary LECs diminished in number, suggesting further functional impairment of the LN by infection. Consistent with this idea, antigen acquisition by LECs, a key function of LN LECs during infection and immunization, was reduced during pathogenic CHIKV infection.

Authors

Cormac J. Lucas, Ryan M. Sheridan, Glennys V. Reynoso, Bennett J. Davenport, Mary K. McCarthy, Aspen Martin, Jay R. Hesselberth, Heather D. Hickman, Beth A.J. Tamburini, Thomas E. Morrison

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Abstract

In autoimmunity, FOXP3+ regulatory T cells (Tregs) skew towards a pro-inflammatory, non-suppressive phenotype and are therefore unable to control the exaggerated autoimmune response. This largely impacts the success of autologous Treg therapy which is currently under investigation for autoimmune diseases, including multiple sclerosis (MS). There is a need to ensure in vivo Treg stability before successful application of Treg therapy. Using genetic fate-mapping mice, we demonstrate that inflammatory, cytokine-expressing exFOXP3 T cells accumulate in the central nervous system during experimental autoimmune encephalomyelitis. In a human in vitro model, we discovered that interaction with inflamed blood-brain barrier endothelial cells (BBB-ECs) induces loss-of-function by Tregs. Transcriptome and cytokine analysis revealed that in vitro migrated Tregs have disrupted regenerative potential, a pro-inflammatory Th1/17 signature and upregulate the mTORC1 signaling pathway. In vitro treatment of migrated human Tregs with the clinically-approved mTORC1 inhibitor rapamycin restored suppression. Finally, flow cytometric analysis indicated an enrichment of inflammatory, less suppressive CD49d+ Tregs in the cerebrospinal fluid of people with MS. In sum, interaction with BBB-ECs is sufficient to affect Treg function, and transmigration triggers an additive pro-inflammatory phenotype switch. These insights help improve the efficacy of autologous Treg therapy of MS.

Authors

Paulien Baeten, Ibrahim Hamad, Cindy Hoeks, Michael Hiltensperger, Bart Van Wijmeersch, Veronica Popescu, Lilian Aly, Veerle Somers, Thomas Korn, Markus Kleinewietfeld, Niels Hellings, Bieke Broux

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Abstract

The efficacy of chimeric antigen receptor (CAR)-T therapy has been limited against brain tumors to date. CAR-T cells infiltrating syngeneic intracerebral SB28-EGFRvIII glioma revealed impaired mitochondrial ATP production and a markedly hypoxic status compared to ones migrating to subcutaneous tumors. Drug screenings to improve metabolic states of T cells under hypoxic conditions led us to evaluate the combination of AMPK activator Metformin and the mTOR inhibitor Rapamycin (Met+Rap). Met+Rap-pretreated mouse CAR-T cells showed activated PPAR-gamma coactivator 1α (PGC-1α) through mTOR inhibition and AMPK activation, and a higher level of mitochondrial spare respiratory capacity than those pretreated with individual drugs or without pretreatment. Moreover, Met+Rap-pretreated CAR-T cells demonstrated persistent and effective anti-glioma cytotoxic activities in the hypoxic condition. Furthermore, a single intravenous infusion of Met+Rap-pretreated CAR-T cells significantly extended the survival of mice bearing intracerebral SB28-EGFRvIII gliomas. Mass cytometric analyses highlighted increased glioma-infiltrating CAR-T cells in the Met+Rap group with fewer Ly6c+ CD11b+ monocytic myeloid-derived suppressor cells in the tumors. Finally, human CAR-T cells pretreated with Met+Rap recapitulated the observations with murine CAR-T cells, demonstrating improved functions in vitro hypoxic conditions. These findings advocate for translational and clinical exploration of Met+Rap-pretreated CAR-T cells in human trials.

Authors

Ryusuke Hatae, Keith Kyewalabye, Akane Yamamichi, Tiffany Chen, Su Phyu, Pavlina Chuntova, Takahide Nejo, Lauren S. Levine, Matthew H. Spitzer, Hideho Okada

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Abstract

Radiotherapy induces a Type I interferon (T1IFN)-mediated anti-tumoral immune response that we hypothesized could be potentiated by a first-in-class ATM inhibitor leading to enhanced innate immune signaling, T1IFN expression, and sensitization to immunotherapy in pancreatic cancer. We evaluated the effects of AZD1390 or a structurally related compound AZD0156 on innate immune signaling and found that both inhibitors enhanced radiation-induced T1IFN expression via the POLIII/RIG-I/MAVS pathway. In immunocompetent syngeneic mouse models of pancreatic cancer, ATM inhibitor enhanced radiation-induced anti-tumoral immune responses and sensitized to anti-PD-L1, producing immunogenic memory and durable tumor control. Therapeutic responses were associated with increased intratumoral CD8+ T cell frequency and effector function. Tumor control was dependent on CD8+ T cells as therapeutic efficacy was blunted in CD8+ T cell-depleted mice. Adaptive immune responses to combination therapy provided systemic control of contralateral tumors outside of the radiation field. Taken together, we show that a clinical candidate ATM inhibitor enhances radiation-induced T1IFN leading to both innate and subsequent adaptive anti-tumoral immune responses and sensitization of otherwise resistant pancreatic cancer to immunotherapy.

Authors

Qiang Zhang, Long Jiang, Weiwei Wang, Amanda K. Huber, Victoria M. Valvo, Kassidy M. Jungles, Erin A. Holcomb, Ashley N. Pearson, Stephanie The, Zhuwen Wang, Leslie A. Parsels, Joshua D. Parsels, Daniel R. Wahl, Arvind Rao, Vaibhav Sahai, Theodore S. Lawrence, Michael D. Green, Meredith A. Morgan

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Abstract

Patients with mutations in the thyroid hormone (TH) cell transporter MCT8 gene develop severe neuro-psychomotor retardation known as the Allan-Herndon-Dudley syndrome (AHDS). It is assumed that this is caused by a reduction in TH signaling in the developing brain, and treatment remains understandably challenging. Given species differences in brain TH transporters and the limitations of studies in mice, we generated brain organoids (BOs) using human iPSCs from MCT8-deficient patients. We found that MCT8-deficient BOs exhibit (i) impaired T3 transport in developing neural cells, as assessed through deiodinase-3-mediated T3 catabolism, (ii) reduced expression of genes involved in neurogenesis and neuronal maturation, and (iii) reduced T3-inducibility of TH-regulated genes. In contrast, the TH-analogs 3,5-diiodothyropropionic acid and 3,3’,5-triiodothyroacetic acid triggered normal responses (induction/repression of T3-responsive genes) in MCT8-deficient BOs, constituting a proof-of-concept that lack of T3 transport underlies the pathophysiology of AHDS, demonstrating the clinical potential for TH analogues to be used in treating AHDS patients. MCT8-deficient BOs represent a species-specific relevant preclinical model that can be utilized to screen drugs with potential benefits as personalized therapeutics for AHDS patients.

Authors

Federico Salas-Lucia, Sergio Escamilla, Antonio C. Bianco, Alexandra Dumitrescu, Samuel Refetoff

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Abstract

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to cystic fibrosis (CF), a life-threating autosomal recessive genetic disease. While recently approved Trikafta dramatically ameliorates CF lung diseases, there is still a lack of effective medicine to treat CF-associated liver disease (CFLD). To address this medical need, we used a recently established CF rabbit model to test if Sotagliflozin, a Sodium-Glucose cotransporter 1 and 2 (SGLT1/2) inhibitor drug that is approved to treat diabetes, can be repurposed to treat CFLD. Sotagliflozin treatment led to systemic benefits to CF rabbits, evidenced by increased appetite and weight gain as well as prolonged lifespan. For CF liver related phenotypes, the animals benefited from normalized blood chemistry and bile acid parameters. Further, Sotagliflozin alleviated non-alcoholic steatohepatitis (NASH)-like phenotypes including liver fibrosis. Intriguingly, Sotagliflozin treatment markedly reduced the otherwise elevated endoplasmic reticulum (ER) stress responses in the liver and other affected organs of CF rabbits. In summary, our work demonstrates that Sotagliflozin attenuates liver disorders in CF rabbits, and merits Sotagliflozin as a potential drug to treat CFLD.

Authors

Xiubin Liang, Xia Hou, Mohamad Bouhamdan, Yifei Sun, Zhenfeng Song, Carthic Rajagopalan, Hong Jiang, Hong-Guang Wei, Jun Song, Dongshan Yang, Yanhong Guo, Yihan Zhang, Hongmei Mou, Jifeng Zhang, Y. Eugene Chen, Fei Sun, Jian-Ping Jin, Kezhong Zhang, Jie Xu

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