Brown adipose tissue (BAT), as the main site of adaptive thermogenesis, exerts beneficial metabolic effects on obesity and insulin resistance. BAT has been previously assumed to contain a homogeneous population of brown adipocytes. Utilizing multiple mouse models capable of genetically labeling different cellular populations, as well as single-cell RNA sequencing and 3D tissue profiling, we discovered a new brown adipocyte subpopulation with low thermogenic activity coexisting with the classical high-thermogenic brown adipocytes within the BAT. Compared with the high-thermogenic brown adipocytes, these low-thermogenic brown adipocytes had substantially lower Ucp1 and Adipoq expression, larger lipid droplets, and lower mitochondrial content. Functional analyses showed that, unlike the high-thermogenic brown adipocytes, the low-thermogenic brown adipocytes have markedly lower basal mitochondrial respiration, and they are specialized in fatty acid uptake. Upon changes in environmental temperature, the 2 brown adipocyte subpopulations underwent dynamic interconversions. Cold exposure converted low-thermogenic brown adipocytes into high-thermogenic cells. A thermoneutral environment had the opposite effect. The recruitment of high-thermogenic brown adipocytes by cold stimulation is not affected by high fat diet feeding, but it does substantially decline with age. Our results revealed a high degree of functional heterogeneity of brown adipocytes.
Anying Song, Wenting Dai, Min Jee Jang, Leonard Medrano, Zhuo Li, Hu Zhao, Mengle Shao, Jiayi Tan, Aimin Li, Tinglu Ning, Marcia M. Miller, Brian Armstrong, Janice M. Huss, Yi Zhu, Yong Liu, Viviana Gradinaru, Xiwei Wu, Lei Jiang, Philipp E. Scherer, Qiong A. Wang
Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40%–50% of adults with CF. The age at onset of CF-related diabetes (CFRD) (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age at onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that 2 common DNA haplotypes formed by the risk variants account for the association with diabetes. Single-cell RNA sequencing (scRNA-Seq) indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells and frequently coexpressed with CF transmembrane conductance regulator (CFTR) along with transcription factors that have binding sites 5′ of SLC26A9. These findings were replicated upon reanalysis of scRNA-Seq data from 4 independent studies. DNA fragments derived from the 5′ region of SLC26A9-bearing variants from the low-risk haplotype generated 12%–20% higher levels of expression in PANC-1 and CFPAC-1 cells compared with the high risk haplotype. Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age at onset of diabetes, suggesting a CFTR-agnostic treatment for a major complication of CF.
Anh-Thu N. Lam, Melis A. Aksit, Briana Vecchio-Pagan, Celeste A. Shelton, Derek L. Osorio, Arianna F. Anzmann, Loyal A. Goff, David C. Whitcomb, Scott M. Blackman, Garry R. Cutting
The c-MYC (MYC) oncoprotein is often overexpressed in human breast cancer; however, its role in driving disease phenotypes is poorly understood. Here, we investigate the role of MYC in HER2+ disease, examining the relationship between HER2 expression and MYC phosphorylation in HER2+ patient tumors and characterizing the functional effects of deregulating MYC expression in the murine NeuNT model of amplified-HER2 breast cancer. Deregulated MYC alone was not tumorigenic, but coexpression with NeuNT resulted in increased MYC Ser62 phosphorylation and accelerated tumorigenesis. The resulting tumors were metastatic and associated with decreased survival compared with NeuNT alone. MYC;NeuNT tumors had increased intertumoral heterogeneity including a subtype of tumors not observed in NeuNT tumors, which showed distinct metaplastic histology and worse survival. The distinct subtypes of MYC;NeuNT tumors match existing subtypes of amplified-HER2, estrogen receptor–negative human tumors by molecular expression, identifying the preclinical utility of this murine model to interrogate subtype-specific differences in amplified-HER2 breast cancer. We show that these subtypes have differential sensitivity to clinical HER2/EGFR–targeted therapeutics, but small-molecule activators of PP2A, the phosphatase that regulates MYC Ser62 phosphorylation, circumvents these subtype-specific differences and ubiquitously suppresses tumor growth, demonstrating the therapeutic utility of this approach in targeting deregulated MYC breast cancers.
Tyler Risom, Xiaoyan Wang, Juan Liang, Xiaoli Zhang, Carl Pelz, Lydia G. Campbell, Jenny Eng, Koei Chin, Caroline Farrington, Goutham Narla, Ellen M. Langer, Xiao-Xin Sun, Yulong Su, Colin J. Daniel, Mu-Shui Dai, Christiane V. Löhr, Rosalie C. Sears
Unconventional T cells that recognize mycobacterial antigens are of great interest as potential vaccine targets against tuberculosis (TB). This includes donor-unrestricted T cells (DURTs), such as mucosa-associated invariant T cells (MAITs), CD1-restricted T cells, and γδ T cells. We exploited the distinctive nature of DURTs and γδ T cell receptors (TCRs) to investigate the involvement of these T cells during TB in the human lung by global TCR sequencing. Making use of surgical lung resections, we investigated the distribution, frequency, and characteristics of TCRs in lung tissue and matched blood from individuals infected with TB. Despite depletion of MAITs and certain CD1-restricted T cells from the blood, we found that the DURT repertoire was well preserved in the lungs, irrespective of disease status or HIV coinfection. The TCRδ repertoire, in contrast, was highly skewed in the lungs, where it was dominated by Vδ1 and distinguished by highly localized clonal expansions, consistent with the nonrecirculating lung-resident γδ T cell population. These data show that repertoire sequencing is a powerful tool for tracking T cell subsets during disease.
Paul Ogongo, Adrie J.C. Steyn, Farina Karim, Kaylesh J. Dullabh, Ismael Awala, Rajhmun Madansein, Alasdair Leslie, Samuel M. Behar
Activation of host T cells that mediate allograft rejection is a 2-step process. The first occurs in secondary lymphoid organs where T cells encounter alloantigens presented by host DCs and differentiate to effectors. Antigen presentation at these sites occurs principally via transfer of intact, donor MHC-peptide complexes from graft cells to host DCs (cross-dressing) or by uptake and processing of donor antigens into allopeptides bound to self-MHC molecules (indirect presentation). The second step takes place in the graft, where effector T cells reengage with host DCs before causing rejection. How host DCs present alloantigens to T cells in the graft is not known. Using mouse islet and kidney transplantation models, imaging cytometry, and 2-photon intravital microscopy, we demonstrate extensive cross-dressing of intragraft host DCs with donor MHC-peptide complexes that occurred early after transplantation, whereas host DCs presenting donor antigen via the indirect pathway were rare. Cross-dressed DCs stably engaged TCR-transgenic effector CD8+ T cells that recognized donor antigen and were sufficient for sustaining acute rejection. In the chronic kidney rejection model, cross-dressing declined over time, but was still conspicuous 8 weeks after transplantation. We conclude that cross-dressing of host DCs with donor MHC molecules is a major antigen presentation pathway driving effector T cell responses within allografts.
Andrew D. Hughes, Daqiang Zhao, Hehua Dai, Khodor I. Abou-Daya, Roger Tieu, Rayan Rammal, Amanda L. Williams, Douglas P. Landsittel, Warren D. Shlomchik, Adrian E. Morelli, Martin H. Oberbarnscheidt, Fadi G. Lakkis
Fibronectin–splice variant containing extra domain A (Fn-EDA) is associated with smooth muscle cells (SMCs) following vascular injury. The role of SMC-derived Fn-EDA in SMC phenotypic switching or its implication in neointimal hyperplasia remains unclear. Herein, using human coronary artery sections with a bare metal stent, we demonstrate the expression of Fn-EDA in the vicinity of SMC-rich neointima and peri-strut areas. In mice, Fn-EDA colocalizes with SMCs in the neointima of injured carotid arteries and promotes neointima formation in the comorbid condition of hyperlipidemia by potentiating SMC proliferation and migration. No sex-based differences were observed. Mechanistic studies suggested that Fn-EDA mediates integrin- and TLR4-dependent proliferation and migration through activation of FAK/Src and Akt1/mTOR signaling, respectively. Specific deletion of Fn-EDA in SMCs, but not in endothelial cells, reduced intimal hyperplasia and suppressed the SMC synthetic phenotype concomitant with decreased Akt1/mTOR signaling. Targeting Fn-EDA in human aortic SMCs suppressed the synthetic phenotype and downregulated Akt1/mTOR signaling. These results reveal that SMC-derived Fn-EDA potentiates phenotypic switching in human and mouse aortic SMCs and neointimal hyperplasia in the mouse. We suggest that targeting Fn-EDA could be explored as a potential therapeutic strategy to reduce neointimal hyperplasia.
Manish Jain, Nirav Dhanesha, Prakash Doddapattar, Mehul R. Chorawala, Manasa K. Nayak, Anne Cornelissen, Liang Guo, Aloke V. Finn, Steven R. Lentz, Anil K. Chauhan
Arcuate nucleus agouti–related peptide (AgRP) neurons play a central role in feeding and are under complex regulation by both homeostatic hormonal and nutrient signals and hypothalamic neuronal pathways. Feeding may also be influenced by environmental cues, sensory inputs, and other behaviors, implying the involvement of higher brain regions. However, whether such pathways modulate feeding through direct synaptic control of AgRP neuron activity is unknown. Here, we show that nociceptin-expressing neurons in the anterior bed nuclei of the stria terminalis (aBNST) make direct GABAergic inputs onto AgRP neurons. We found that activation of these neurons inhibited AgRP neurons and feeding. The activity of these neurons increased upon food availability, and their ablation resulted in obesity. Furthermore, these neurons received afferent inputs from a range of upstream brain regions as well as hypothalamic nuclei. Therefore, aBNST GABAergic nociceptin neurons may act as a gateway to feeding behavior by connecting AgRP neurons to both homeostatic and nonhomeostatic neuronal inputs.
Mark A. Smith, Agharul I. Choudhury, Justyna A. Glegola, Paulius Viskaitis, Elaine E. Irvine, Pedro Caldas Custodio de Campos Silva, Sanjay Khadayate, Hanns Ulrich Zeilhofer, Dominic J. Withers
Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single-strand binding protein (SSBP1) in 4 families with dominant and 1 with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect the amount of SSBP1 protein and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids, and 7S-DNA amounts as well as mtDNA replication, affecting replisome machinery. The variable mtDNA depletion in cells was reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits, and complex amount and assembly. mtDNA depletion and cytochrome c oxidase–negative cells were found ex vivo in biopsies of affected tissues, such as kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by WT mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as a cause of human pathology.
Valentina Del Dotto, Farid Ullah, Ivano Di Meo, Pamela Magini, Mirjana Gusic, Alessandra Maresca, Leonardo Caporali, Flavia Palombo, Francesca Tagliavini, Evan Harris Baugh, Bertil Macao, Zsolt Szilagyi, Camille Peron, Margaret A. Gustafson, Kamal Khan, Chiara La Morgia, Piero Barboni, Michele Carbonelli, Maria Lucia Valentino, Rocco Liguori, Vandana Shashi, Jennifer Sullivan, Shashi Nagaraj, Mays El-Dairi, Alessandro Iannaccone, Ioana Cutcutache, Enrico Bertini, Rosalba Carrozzo, Francesco Emma, Francesca Diomedi-Camassei, Claudia Zanna, Martin Armstrong, Matthew Page, Nicholas Stong, Sylvia Boesch, Robert Kopajtich, Saskia Wortmann, Wolfgang Sperl, Erica E. Davis, William C. Copeland, Marco Seri, Maria Falkenberg, Holger Prokisch, Nicholas Katsanis, Valeria Tiranti, Tommaso Pippucci, Valerio Carelli
Currently, an effective targeted therapy for pancreatitis is lacking. Hereditary pancreatitis (HP) is a heritable, autosomal-dominant disorder with recurrent acute pancreatitis (AP) progressing to chronic pancreatitis (CP) and a markedly increased risk of pancreatic cancer. In 1996, mutations in PRSS1 were linked to the development of HP. Here, we developed a mouse model by inserting a full-length human PRSS1R122H gene, the most commonly mutated gene in human HP, into mice. Expression of PRSS1R122H protein in the pancreas markedly increased stress signaling pathways and exacerbated AP. After the attack of AP, all PRSS1R122H mice had disease progression to CP, with similar histologic features as those observed in human HP. By comparing PRSS1R122H mice with PRSS1WT mice as well as enzymatically inactivated Dead-PRSS1R122H mice, we unraveled that increased trypsin activity is the mechanism for R122H mutation to sensitize mice to the development of pancreatitis. We further discovered that trypsin inhibition, in combination with anticoagulation therapy, synergistically prevented progression to CP in PRSS1R122H mice. These animal models help us better understand the complex nature of this disease and provide powerful tools for developing and testing novel therapeutics for human pancreatitis.
Fu Gui, Yuebo Zhang, Jianhua Wan, Xianbao Zhan, Yao Yao, Yinghua Li, Ashley N. Haddock, Ji Shi, Jia Guo, Jiaxiang Chen, Xiaohui Zhu, Brandy H. Edenfield, Lu Zhuang, Cheng Hu, Ying Wang, Debabrata Mukhopadhyay, Evette S. Radisky, Lizhi Zhang, Aurelia Lugea, Stephen J. Pandol, Yan Bi, Baoan Ji
Whether respiratory epithelial cells regulate the final transit of extravasated neutrophils into the inflamed airspace or are a passive barrier is poorly understood. Alveolar epithelial type 1 (AT1) cells, best known for solute transport and gas exchange, have few established immune roles. Epithelial membrane protein 2 (EMP2), a tetraspan protein that promotes recruitment of integrins to lipid rafts, is highly expressed in AT1 cells, but has no known function in lung biology. Here, we show that Emp2–/– mice exhibit reduced neutrophil influx into the airspace after a wide range of inhaled exposures. During bacterial pneumonia, Emp2–/– mice had attenuated neutrophilic lung injury and improved survival. Bone marrow chimeras, intravital neutrophil labeling, and in vitro assays suggested that defective transepithelial migration of neutrophils into the alveolar lumen occurs in Emp2–/– lungs. Emp2–/– AT1 cells had dysregulated surface display of multiple adhesion molecules, associated with reduced raft abundance. Epithelial raft abundance was dependent upon putative cholesterol-binding motifs in EMP2, whereas EMP2 supported adhesion molecule display and neutrophil transmigration through suppression of caveolins. Taken together, we propose that EMP2-dependent membrane organization ensures proper display on AT1 cells of a suite of proteins required to instruct paracellular neutrophil traffic into the alveolus.
Wan-Chi Lin, Kymberly M. Gowdy, Jennifer H. Madenspacher, Rachel L. Zemans, Kazuko Yamamoto, Miranda Lyons-Cohen, Hideki Nakano, Kyathanahalli Janardhan, Carmen J. Williams, Donald N. Cook, Joseph P. Mizgerd, Michael B. Fessler
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