Issue published June 15, 2021 Previous issue

On the cover: Down syndrome patient iPSC–derived cerebral organoids

In this issue, Tang et al. characterize cerebral organoid–derived induced pluripotent stem cells from individuals with trisomy 21 to explore cell type–specific changes associated with Down syndrome. The cover image shows immunostaining for the neural progenitor marker SOX1 (red), doublecortin (green), and the mitotic marker phosphorylated histone H3 (gray); and Hoechst staining of nuclei (blue) in trisomy 21 organoids.

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Review Series
Abstract

Traumatic brain injury (TBI) is a chronic and progressive disease, and management requires an understanding of both the primary neurological injury and the secondary sequelae that affect peripheral organs, including the gastrointestinal (GI) tract. The brain-gut axis is composed of bidirectional pathways through which TBI-induced neuroinflammation and neurodegeneration impact gut function. The resulting TBI-induced dysautonomia and systemic inflammation contribute to the secondary GI events, including dysmotility and increased mucosal permeability. These effects shape, and are shaped by, changes in microbiota composition and activation of resident and recruited immune cells. Microbial products and immune cell mediators in turn modulate brain-gut activity. Importantly, secondary enteric inflammatory challenges prolong systemic inflammation and worsen TBI-induced neuropathology and neurobehavioral deficits. The importance of brain-gut communication in maintaining GI homeostasis highlights it as a viable therapeutic target for TBI. Currently, treatments directed toward dysautonomia, dysbiosis, and/or systemic inflammation offer the most promise.

Authors

Marie Hanscom, David J. Loane, Terez Shea-Donohue

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Reviews
Abstract

B cells have a prominent role in the pathogenesis of systemic lupus erythematosus (SLE). They are mediators of inflammation through the production of pathogenic antibodies that augment inflammation and cause direct tissue and cell damage. Multiple therapeutic agents targeting B cells have been successfully used in mouse models of SLE; however, these preclinical studies have led to approval of only one new agent to treat patients with SLE: belimumab, a monoclonal antibody targeting B cell–activating factor (BAFF). Integrating the experience acquired from previous clinical trials with the knowledge generated by new studies about mechanisms of B cell contributions to SLE in specific groups of patients is critical to the development of new treatment strategies that will help to improve outcomes in patients with SLE. In particular, a sharper focus on B cell differentiation to plasma cells is warranted.

Authors

Yemil Atisha-Fregoso, Bahtiyar Toz, Betty Diamond

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Abstract

As a result of impressive increases in our knowledge of rodent and human immunology, the understanding of the pathophysiologic mechanisms underlying graft-versus-host disease (GVHD) has dramatically improved in the past 15 years. Despite improved knowledge, translation to clinical care has not proceeded rapidly, and results from experimental models have been inconsistent in their ability to predict the clinical utility of new therapeutic agents. In parallel, new tools in immunology have allowed in-depth analyses of the human system and have recently been applied in the field of clinical GVHD. Notwithstanding these advances, there is a relative paucity of mechanistic insights into human translational research, and this remains an area of high unmet need. Here we review selected recent advances in both preclinical experimental transplantation and translational human studies, including new insights into human immunology, the microbiome, and regenerative medicine. We focus on the fact that both approaches can interactively improve our understanding of both acute and chronic GVHD biology and open the door to improved therapeutics and successes.

Authors

Gérard Socié, Leslie S. Kean, Robert Zeiser, Bruce R. Blazar

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Commentaries
Abstract

During progression to both types 1 and 2 diabetes (T1D, T2D), there is a striking loss of glucose-induced first-phase insulin release (FPIR), which is known to predict the onset of T1D. The contribution of reduced β cell mass to the onset of hyperglycemia remains unclear. In this issue of the JCI, Mezza et al. report on their study of patients with pancreatic neoplasms before and after partial pancreatectomy to evaluate the impact of reduced β cell mass on the development of diabetes. The authors found that reduced FPIR predicted diabetes when 50% of the pancreas was removed. These findings suggest that low or absent FPIR indicates that β cell mass can no longer compensate for increased insulin needs. Notably, clinicians may use reduction of FPIR as a warning that progression to T2D is underway.

Authors

Gordon C. Weir, Susan Bonner-Weir

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Abstract

Efforts to best protect the world from SARS-CoV-2 as variants emerge and despite limited vaccine supply are ongoing. One strategy that may maximize vaccine coverage and expedite immunization campaigns involves providing single mRNA vaccine doses to individuals with previous COVID-19. In this issue of the JCI, two independent studies, one by Levi and Azzolini et al. and another by Mazzoni and Di Lauria et al., explored vaccine responses in individuals previously infected with the virus. Levi and Azzolini and colleagues used multilinear regression models to correlate exposure and symptoms with antibody response to the vaccine. Mazzoni and Di Lauria and colleagues characterized B cell and T cell kinetics in whole blood after one and two doses of vaccine in health care workers with and without previous infection. Both studies indicated that one vaccine dose may sufficiently protect individuals who have recovered from COVID-19. Implementing a single-dose mRNA vaccine protocol in previously symptomatic individuals may facilitate and expedite immunization campaigns.

Authors

Gonzalo Perez Marc, Damian Alvarez-Paggi, Fernando P. Polack

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Abstract

Immune-mediated kidney diseases are a leading cause of end-stage renal disease. Despite recent discoveries, the immunopathogenesis of this heterogeneous disease group remains incompletely understood, which is a major reason for the lack of specific therapies and targeted interventions. Accumulating evidence suggests that cytokines related to the T cell response play an important role in renal autoimmunity. In this issue of the JCI, Li et al. demonstrate that IL-23 directly regulates the metabolism of parenchymal kidney cells, thereby generating a proinflammatory microenvironment that exacerbates T cell–driven renal tissue damage. These findings identify the IL-23/IL-17 axis as a key mediator of renal tissue injury and open new avenues for the development of pathogenesis-based treatment strategies in renal inflammatory diseases.

Authors

Christian F. Krebs, Jan-Eric Turner, Jan-Hendrik Riedel, Ulf Panzer

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Research Articles
Abstract

Down syndrome (DS), caused by trisomy of chromosome 21, occurs in 1 of every 800 live births. Early defects in cortical development likely account for the cognitive impairments in DS, although the underlying molecular mechanism remains elusive. Here, we performed histological assays and unbiased single-cell RNA-Seq (scRNA-Seq) analysis on cerebral organoids derived from 4 euploid cell lines and from induced pluripotent stem cells (iPSCs) from 3 individuals with trisomy 21 to explore cell-type–specific abnormalities associated with DS during early brain development. We found that neurogenesis was significantly affected, given the diminished proliferation and decreased expression of layer II and IV markers in cortical neurons in the subcortical regions; this may have been responsible for the reduced size of the organoids. Furthermore, suppression of the DSCAM/PAK1 pathway, which showed enhanced activity in DS, using CRISPR/Cas9, CRISPR interference (CRISPRi), or small-molecule inhibitor treatment reversed abnormal neurogenesis, thereby increasing the size of organoids derived from DS iPSCs. Our study demonstrates that 3D cortical organoids developed in vitro are a valuable model of DS and provide a direct link between dysregulation of the DSCAM/PAK1 pathway and developmental brain defects in DS.

Authors

Xiao-Yan Tang, Lei Xu, Jingshen Wang, Yuan Hong, Yuanyuan Wang, Qian Zhu, Da Wang, Xin-Yue Zhang, Chun-Yue Liu, Kai-Heng Fang, Xiao Han, Shihua Wang, Xin Wang, Min Xu, Anita Bhattacharyya, Xing Guo, Mingyan Lin, Yan Liu

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Abstract

Severe asthma remains challenging to manage and has limited treatment options. We have previously shown that targeting smooth muscle integrin α5β1 interaction with fibronectin can mitigate the effects of airway hyperresponsiveness by impairing force transmission. In this study, we show that another member of the integrin superfamily, integrin α2β1, is present in airway smooth muscle and capable of regulating force transmission via cellular tethering to the matrix protein collagen I and, to a lesser degree, laminin-111. The addition of an inhibitor of integrin α2β1 impaired IL-13–enhanced contraction in mouse tracheal rings and human bronchial rings and abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. We confirmed that this effect was not due to alterations in classic intracellular myosin light chain phosphorylation regulating muscle shortening. Although IL-13 did not affect surface expression of α2β1, it did increase α2β1-mediated adhesion and the level of expression of an activation-specific epitope on the β1 subunit. We developed a method to simultaneously quantify airway narrowing and muscle shortening using 2-photon microscopy and demonstrated that inhibition of α2β1 mitigated IL-13–enhanced airway narrowing without altering muscle shortening by impairing the tethering of muscle to the surrounding matrix. Our data identified cell matrix tethering as an attractive therapeutic target to mitigate the severity of airway contraction in asthma.

Authors

Sean Liu, Uyen Ngo, Xin-Zi Tang, Xin Ren, Wenli Qiu, Xiaozhu Huang, William DeGrado, Christopher D.C. Allen, Hyunil Jo, Dean Sheppard, Aparna B. Sundaram

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Abstract

Cutaneous melanoma remains the most lethal skin cancer, and ranks third among all malignancies in terms of years of life lost. Despite the advent of immune checkpoint and targeted therapies, only roughly half of patients with advanced melanoma achieve a durable remission. Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that regulates metabolism and other biological processes. Germline Sirt5 deficiency is associated with mild phenotypes in mice. Here we showed that SIRT5 was required for proliferation and survival across all cutaneous melanoma genotypes tested, as well as uveal melanoma, a genetically distinct melanoma subtype that arises in the eye and is incurable once metastatic. Likewise, SIRT5 was required for efficient tumor formation by melanoma xenografts and in an autochthonous mouse Braf Pten–driven melanoma model. Via metabolite and transcriptomic analyses, we found that SIRT5 was required to maintain histone acetylation and methylation levels in melanoma cells, thereby promoting proper gene expression. SIRT5-dependent genes notably included MITF, a key lineage-specific survival oncogene in melanoma, and the c-MYC proto-oncogene. SIRT5 may represent a druggable genotype-independent addiction in melanoma.

Authors

William Giblin, Lauren Bringman-Rodenbarger, Angela H. Guo, Surinder Kumar, Alexander C. Monovich, Ahmed M. Mostafa, Mary E. Skinner, Michelle Azar, Ahmed S.A. Mady, Carolina H. Chung, Namrata Kadambi, Keith-Allen Melong, Ho-Joon Lee, Li Zhang, Peter Sajjakulnukit, Sophie Trefely, Erika L. Varner, Sowmya Iyer, Min Wang, James S. Wilmott, H. Peter Soyer, Richard A. Sturm, Antonia L. Pritchard, Aleodor A. Andea, Richard A. Scolyer, Mitchell S. Stark, David A. Scott, Douglas R. Fullen, Marcus W. Bosenberg, Sriram Chandrasekaran, Zaneta Nikolovska-Coleska, Monique E. Verhaegen, Nathaniel W. Snyder, Miguel N. Rivera, Andrei L. Osterman, Costas A. Lyssiotis, David B. Lombard

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Abstract

Therapies targeting VEGF have proven only modestly effective for the treatment of proliferative sickle cell retinopathy (PSR), the leading cause of blindness in patients with sickle cell disease. Here, we shift our attention upstream from the genes that promote retinal neovascularization (NV) to the transcription factors that regulate their expression. We demonstrated increased expression of HIF-1α and HIF-2α in the ischemic inner retina of PSR eyes. Although both HIFs participated in promoting VEGF expression by hypoxic retinal Müller cells, HIF-1 alone was sufficient to promote retinal NV in mice, suggesting that therapies targeting only HIF-2 would not be adequate to prevent PSR. Nonetheless, administration of a HIF-2–specific inhibitor currently in clinical trials (PT2385) inhibited NV in the oxygen-induced retinopathy (OIR) mouse model. To unravel these discordant observations, we examined the expression of HIFs in OIR mice and demonstrated rapid but transient accumulation of HIF-1α but delayed and sustained accumulation of HIF-2α; simultaneous expression of HIF-1α and HIF-2α was not observed. Staggered HIF expression was corroborated in hypoxic adult mouse retinal explants but not in human retinal organoids, suggesting that this phenomenon may be unique to mice. Using pharmacological inhibition or an in vivo nanoparticle-mediated RNAi approach, we demonstrated that inhibiting either HIF was effective for preventing NV in OIR mice. Collectively, these results explain why inhibition of either HIF-1α or HIF-2α is equally effective for preventing retinal NV in mice but suggest that therapies targeting both HIFs will be necessary to prevent NV in patients with PSR.

Authors

Jing Zhang, Yaowu Qin, Mireya Martinez, Miguel Flores-Bellver, Murilo Rodrigues, Aumreetam Dinabandhu, Xuan Cao, Monika Deshpande, Yu Qin, Silvia Aparicio-Domingo, Yuan Rui, Stephany Y. Tzeng, Shaima Salman, Jin Yuan, Adrienne W. Scott, Jordan J. Green, M. Valeria Canto-Soler, Gregg L. Semenza, Silvia Montaner, Akrit Sodhi

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Abstract

Chronic cellular stress associated with neurodegenerative disease can result in the persistence of stress granule (SG) structures, membraneless organelles that form in response to cellular stress. In Huntington’s disease (HD), chronic expression of mutant huntingtin generates various forms of cellular stress, including activation of the unfolded protein response and oxidative stress. However, it has yet to be determined whether SGs are a feature of HD neuropathology. We examined the miRNA composition of extracellular vesicles (EVs) present in the cerebrospinal fluid (CSF) of patients with HD and show that a subset of their target mRNAs were differentially expressed in the prefrontal cortex. Of these targets, SG components were enriched, including the SG-nucleating Ras GTPase-activating protein-binding protein 1 (G3BP1). We investigated localization and levels of G3BP1 and found a significant increase in the density of G3BP1-positive granules in the cortex and hippocampus of R6/2 transgenic mice and in the superior frontal cortex of the brains of patients with HD. Intriguingly, we also observed that the SG-associated TAR DNA-binding protein 43 (TDP43), a nuclear RNA/DNA binding protein, was mislocalized to the cytoplasm of G3BP1 granule–positive HD cortical neurons. These findings suggest that G3BP1 SG dynamics may play a role in the pathophysiology of HD.

Authors

Isabella I. Sanchez, Thai B. Nguyen, Whitney E. England, Ryan G. Lim, Anthony Q. Vu, Ricardo Miramontes, Lauren M. Byrne, Sebastian Markmiller, Alice L. Lau, Iliana Orellana, Maurice A. Curtis, Richard Lewis Maxwell Faull, Gene W. Yeo, Christie D. Fowler, Jack C. Reidling, Edward J. Wild, Robert C. Spitale, Leslie M. Thompson

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Abstract

FOXP3+ Tregs are expanded within the inflamed intestine of human Crohn’s disease, yet FOXP3-mediated gene repression within these cells is lost. The polycomb repressive complexes play a role in FOXP3 target gene regulation, but deeper mechanistic insight is incomplete. We have now specifically identified the polycomb-repressive complex 1 (PRC1) family member, BMI1 in the regulation of a proinflammatory enhancer network in both human and murine Tregs. Using human Tregs and lamina propria T cells, we inferred PRC1 to regulate Crohn’s associated gene networks through assays of chromatin accessibility. Conditional deletion of BMI1 in murine FOXP3+ cells led to systemic inflammation. BMI1-deficient Tregs beared a TH1/TH17-like phenotype as assessed by assays of genome wide transcription, chromatin accessibility and proteomic techniques. Finally, BMI1 mutant FOXP3+ cells did not suppress colitis in the adoptive transfer model of human inflammatory bowel disease. We propose that BMI1 plays an important role in enforcing Treg identity in vitro and in vivo. Loss of Treg identity via genetic or transient BMI1 depletion perturbs the epigenome and converts Tregs into Th1/Th17-like proinflammatory cells, a transition relevant to human Crohn’s disease associated CD4+ T cells.

Authors

Michelle M. Gonzalez, Adebowale O. Bamidele, Phyllis A. Svingen, Mary R. Sagstetter, Thomas C. Smyrk, Joseph M. Gaballa, Feda H. Hamdan, Robyn Laura Kosinsky, Hunter R. Gibbons, Zhifu Sun, Zhenqing Ye, Asha Nair, Guilherme P. Ramos, Manuel B. Braga Neto, Alexander Q. Wixom, Angela J. Mathison, Steven A. Johnsen, Raul Urrutia, William A. Faubion Jr.

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Abstract

Cancer cells reprogram lipid metabolism during their malignant progression, but limited information is currently available on the involvement of alterations in fatty acid synthesis in cancer development. We herein demonstrate that acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme for fatty acid synthesis, plays a critical role in regulating the growth and differentiation of leukemia-initiating cells. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPA, a transcription factor regulating myeloid differentiation, for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). We identified ACC1 as a target of the Trib1-COP1 complex and found that an ACC1 mutant resistant to degradation because of the lack of a Trib1-binding site attenuated complex-driven leukemogenesis. Stable ACC1 protein expression suppressed the growth-promoting activity and increased ROS levels with the consumption of NADPH in a primary bone marrow culture, and delayed the onset of AML with increases in mature myeloid cells in mouse models. ACC1 promoted the terminal differentiation of Trib1-COP1–expressing cells and eradicated leukemia-initiating cells in the early phase of leukemic progression. These results indicate that ACC1 is a natural inhibitor of AML development. The upregulated expression of the ACC1 protein has potential as an effective strategy for cancer therapy.

Authors

Hidenori Ito, Ikuko Nakamae, Jun-ya Kato, Noriko Yoneda-Kato

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Abstract

Interstitial kidney inflammation is present in various nephritides in which serum interleukin 23 (IL-23) is elevated. Here we showed that murine and human renal tubular epithelial cells (TECs) expressing the IL-23 receptor (IL-23R) responded to IL-23 by inducing intracellular calcium flux, enhancing glycolysis, and upregulating calcium/calmodulin kinase IV (CaMK4), which resulted in suppression of the expression of the arginine-degrading enzyme arginase 1 (ARG1), thus increasing in situ levels of free L-arginine. Limited availability of arginine suppressed the ability of infiltrating T cells to proliferate and produce inflammatory cytokines. TECs from humans and mice with nephritis expressed increased levels of IL-23R and CaMK4 but reduced levels of ARG1. TEC-specific deletion of Il23r or Camk4 suppressed inflammation, whereas deletion of Arg1 exacerbated inflammation in different murine disease models. Finally, TEC-specific delivery of a CaMK4 inhibitor specifically curbed renal inflammation in lupus-prone mice without affecting systemic inflammation. Our data offer the first evidence to our knowledge of the immunosuppressive capacity of TECs through a mechanism that involves competitive uptake of arginine and signify the importance of modulation of an inflammatory cytokine in the function of nonlymphoid cells, which leads to the establishment of an inflammatory microenvironment. New approaches to treat kidney inflammation should consider restoring the immunosuppressive capacity of TECs.

Authors

Hao Li, Maria G. Tsokos, Rhea Bhargava, Iannis E. Adamopoulos, Hanni Menn-Josephy, Isaac E. Stillman, Philip Rosenstiel, Jarrat Jordan, George C. Tsokos

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Abstract

BACKGROUND Deciphering the function of the many genes previously classified as uncharacterized open reading frame (ORF) would complete our understanding of a cell’s function and its pathophysiology.METHODS Whole-exome sequencing, yeast 2-hybrid and transcriptome analyses, and molecular characterization were performed in this study to uncover the function of the C2orf69 gene.RESULTS We identified loss-of-function mutations in the uncharacterized C2orf69 gene in 8 individuals with brain abnormalities involving hypomyelination and microcephaly, liver dysfunction, and recurrent autoinflammation. C2orf69 contains an N-terminal signal peptide that is required and sufficient for mitochondrial localization. Consistent with mitochondrial dysfunction, the patients showed signs of respiratory chain defects, and a CRISPR/Cas9-KO cell model of C2orf69 had similar respiratory chain defects. Patient-derived cells revealed alterations in immunological signaling pathways. Deposits of periodic acid–Schiff–positive (PAS-positive) material in tissues from affected individuals, together with decreased glycogen branching enzyme 1 (GBE1) activity, indicated an additional impact of C2orf69 on glycogen metabolism.CONCLUSIONS Our study identifies C2orf69 as an important regulator of human mitochondrial function and suggests that this gene has additional influence on other metabolic pathways.

Authors

Eva Lausberg, Sebastian Gießelmann, Joseph P. Dewulf, Elsa Wiame, Anja Holz, Ramona Salvarinova, Clara D. van Karnebeek, Patricia Klemm, Kim Ohl, Michael Mull, Till Braunschweig, Joachim Weis, Clemens J. Sommer, Stephanie Demuth, Claudia Haase, Claudia Stollbrink-Peschgens, François-Guillaume Debray, Cecile Libioulle, Daniela Choukair, Prasad T. Oommen, Arndt Borkhardt, Harald Surowy, Dagmar Wieczorek, Norbert Wagner, Robert Meyer, Thomas Eggermann, Matthias Begemann, Emile Van Schaftingen, Martin Häusler, Klaus Tenbrock, Lambert van den Heuvel, Miriam Elbracht, Ingo Kurth, Florian Kraft

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Abstract

Hypoxia is a hallmark of solid tumors that promotes cell growth, survival, and metastasis and confers resistance to chemo and radiotherapies. Hypoxic responses are largely mediated by the transcription factors hypoxia-inducible factor 1α (HIF-1α) and HIF-2α. Our work demonstrates that HIF-2α is essential for colorectal cancer (CRC) progression. However, targeting hypoxic cells is difficult, and tumors rapidly acquire resistance to inhibitors of HIF-2α. To overcome this limitation, we performed a small molecule screen to identify HIF-2α–dependent vulnerabilities. Several known ferroptosis activators and dimethyl fumarate (DMF), a cell-permeable mitochondrial metabolite derivative, led to selective synthetic lethality in HIF-2α–expressing tumor enteroids. Our work demonstrated that HIF-2α integrated 2 independent forms of cell death via regulation of cellular iron and oxidation. First, activation of HIF-2α upregulated lipid and iron regulatory genes in CRC cells and colon tumors in mice and led to a ferroptosis-susceptible cell state. Second, via an iron-dependent, lipid peroxidation–independent pathway, HIF-2α activation potentiated ROS via irreversible cysteine oxidation and enhanced cell death. Inhibition or knockdown of HIF-2α decreased ROS and resistance to oxidative cell death in vitro and in vivo. Our results demonstrated a mechanistic vulnerability in cancer cells that were dependent on HIF-2α that can be leveraged for CRC treatment.

Authors

Rashi Singhal, Sreedhar R. Mitta, Nupur K. Das, Samuel A. Kerk, Peter Sajjakulnukit, Sumeet Solanki, Anthony Andren, Roshan Kumar, Kenneth P. Olive, Ruma Banerjee, Costas A. Lyssiotis, Yatrik M. Shah

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Abstract

Although tissue uptake of fatty acids from chylomicrons is primarily via lipoprotein lipase (LpL) hydrolysis of triglycerides (TGs), studies of patients with genetic LpL deficiency suggest additional pathways deliver dietary lipids to tissues. Despite an intact endothelial cell (EC) barrier, hyperchylomicronemic patients accumulate chylomicron-derived lipids within skin macrophages, leading to the clinical finding eruptive xanthomas. We explored whether an LpL-independent pathway exists for transfer of circulating lipids across the EC barrier. We found that LpL-deficient mice had a marked increase in aortic EC lipid droplets before and after a fat gavage. Cultured ECs internalized chylomicrons, which were hydrolyzed within lysosomes. The products of this hydrolysis fueled lipid droplet biogenesis in ECs and triggered lipid accumulation in cocultured macrophages. EC chylomicron uptake was inhibited by competition with HDL and knockdown of the scavenger receptor-BI (SR-BI). In vivo, SR-BI knockdown reduced TG accumulation in aortic ECs and skin macrophages of LpL-deficient mice. Thus, ECs internalize chylomicrons, metabolize them in lysosomes, and either store or release their lipids. This latter process may allow accumulation of TGs within skin macrophages and illustrates a pathway that might be responsible for creation of eruptive xanthomas.

Authors

Ainara G. Cabodevilla, Songtao Tang, Sungwoon Lee, Adam E. Mullick, Jose O. Aleman, M. Mahmood Hussain, William C. Sessa, Nada A. Abumrad, Ira J. Goldberg

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Abstract

Melanoma dedifferentiation has been reported to be a state of cellular resistance to targeted therapies and immunotherapies as cancer cells revert to a more primitive cellular phenotype. Here, we show that, counterintuitively, the biopsies of patient tumors that responded to anti–programmed cell death 1 (anti–PD-1) therapy had decreased expression of melanocytic markers and increased neural crest markers, suggesting treatment-induced dedifferentiation. When modeling the effects in vitro, we documented that melanoma cell lines that were originally differentiated underwent a process of neural crest dedifferentiation when continuously exposed to IFN-γ, through global chromatin landscape changes that led to enrichment in specific hyperaccessible chromatin regions. The IFN-γ–induced dedifferentiation signature corresponded with improved outcomes in patients with melanoma, challenging the notion that neural crest dedifferentiation is entirely an adverse phenotype.

Authors

Yeon Joo Kim, Katherine M. Sheu, Jennifer Tsoi, Gabriel Abril-Rodriguez, Egmidio Medina, Catherine S. Grasso, Davis Y. Torrejon, Ameya S. Champhekar, Kevin Litchfield, Charles Swanton, Daniel E. Speiser, Philip O. Scumpia, Alexander Hoffmann, Thomas G. Graeber, Cristina Puig-Saus, Antoni Ribas

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Abstract

The discovery of embryonic cell–free DNA (cfDNA) in spent embryo culture media (SECM) has brought hope for noninvasive preimplantation genetic testing. However, the cellular origins of SECM cfDNA are not sufficiently understood, and methods for determining maternal DNA contamination are limited. Here, we performed whole-genome DNA methylation sequencing for SECM cfDNA. Our results demonstrated that SECM cfDNA was derived from blastocysts, cumulus cells, and polar bodies. We identified the cumulus-specific differentially methylated regions (DMRs) and oocyte/polar body–specific DMRs, and established an algorithm for deducing the cumulus, polar body, and net maternal DNA contamination ratios in SECM. We showed that DNA methylation sequencing accurately detected chromosome aneuploidy in SECM and distinguished SECM samples with low and high false negative rates and gender discordance rates, after integrating the origin analysis. Our work provides insights into the characterization of embryonic DNA in SECM and provides a perspective for noninvasive preimplantation genetic testing in reproductive medicine.

Authors

Yidong Chen, Yuan Gao, Jialin Jia, Liang Chang, Ping Liu, Jie Qiao, Fuchou Tang, Lu Wen, Jin Huang

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Abstract

Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor (GLP-1R) agonist, delivered superior glycemic control and weight loss compared with GLP-1R agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine whether GIPR agonism contributes, we compared the effect of tirzepatide in obese WT and Glp-1r–null mice. In the absence of GLP-1R–induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support of this, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino acids (BCAAs) and ketoacids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid, and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual-receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.

Authors

Ricardo J. Samms, Michael E. Christe, Kyla A.L. Collins, Valentina Pirro, Brian A. Droz, Adrienne K. Holland, Jessica L. Friedrich, Samantha Wojnicki, Debra L. Konkol, Richard Cosgrove, Ellen P.S. Conceição Furber, Xiaoping Ruan, Libbey S. O’Farrell, Annie M. Long, Mridula Dogra, Jill A. Willency, Yanzhu Lin, Liyun Ding, Christine C. Cheng, Over Cabrera, Daniel A. Briere, Jorge Alsina-Fernandez, Ruth E. Gimeno, Julie S. Moyers, Tamer Coskun, Matthew P. Coghlan, Kyle W. Sloop, William C. Roell

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Abstract

Sepsis survivors exhibit impaired responsiveness to antigen (Ag) challenge associated with increased mortality from infection. The contribution of follicular dendritic cells (FDCs) in the impaired humoral response in sepsis-surviving mice is investigated in this study. We demonstrated that mice subjected to sepsis from cecal ligation and puncture (CLP mice) have reduced NP-specific high-affinity class-switched Ig antibodies (Abs) compared with sham-operated control mice following immunization with the T cell–dependent Ag, NP-CGG. NP-specific germinal center (GC) B cells in CLP mice exhibited reduced TNF-α and AID mRNA expression compared with sham-operated mice. CLP mice showed a reduction in FDC clusters, a reduced binding of immune complexes on FDCs, and reduced mRNA expression of CR2, ICAM-1, VCAM-1, FcγRIIB, TNFR1, IKK2, and LTβR compared with sham-operated mice. Adoptive transfer studies showed that there was no B cell–intrinsic defect. In summary, our data suggest that the reduced Ag-specific Ab response in CLP mice is secondary to a disruption in FDC and GC B cell function.

Authors

Minakshi Rana, Andrea La Bella, Rivka Lederman, Bruce T. Volpe, Barbara Sherry, Betty Diamond

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Abstract

BACKGROUND The appearance of hyperglycemia is due to insulin resistance, functional deficits in the secretion of insulin, and a reduction of β cell mass. There is a long-standing debate as to the relative contribution of these factors to clinically manifesting β cell dysfunction. The aim of this study was to verify the acute effect of one of these factors, the reduction of β cell mass, on the subsequent development of hyperglycemia.METHODS To pursue this aim, nondiabetic patients, scheduled for identical pancreaticoduodenectomy surgery, underwent oral glucose tolerance tests (OGTT) and hyperglycemic clamp (HC) procedures, followed by arginine stimulation before and after surgery. Based on postsurgery OGTT, subjects were divided into 3 groups depending on glucose tolerance: normal glucose tolerance (post-NGT), impaired glucose tolerance (post-IGT), or having diabetes mellitus (post-DM).RESULTS At baseline, the 3 groups showed similar fasting glucose and insulin levels; however, examining the various parameters, we found that reduced first-phase insulin secretion, reduced glucose sensitivity, and rate sensitivity were predictors of eventual postsurgery development of IGT and diabetes.CONCLUSION Despite comparable functional mass and fasting glucose and insulin levels at baseline and the very same 50% mass reduction, only reduced first-phase insulin secretion and glucose sensitivity predicted the appearance of hyperglycemia. These functional alterations could be pivotal to the pathogenesis of type 2 diabetes (T2DM).TRIAL REGISTRATION ClinicalTrials.gov NCT02175459.FUNDING Università Cattolica del Sacro Cuore; Italian Ministry of Education, University and Research; European Foundation for the Study of Diabetes.

Authors

Teresa Mezza, Pietro Manuel Ferraro, Gianfranco Di Giuseppe, Simona Moffa, Chiara M.A. Cefalo, Francesca Cinti, Flavia Impronta, Umberto Capece, Giuseppe Quero, Alfredo Pontecorvi, Andrea Mari, Sergio Alfieri, Andrea Giaccari

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Abstract

T cells are involved in control of coronavirus disease 2019 (COVID-19), but limited knowledge is available on the relationship between antigen-specific T cell response and disease severity. Here, we used flow cytometry to assess the magnitude, function, and phenotype of SARS coronavirus 2–specific (SARS-CoV-2–specific) CD4+ T cells in 95 hospitalized COVID-19 patients, 38 of them being HIV-1 and/or tuberculosis (TB) coinfected, and 38 non–COVID-19 patients. We showed that SARS-CoV-2–specific CD4+ T cell attributes, rather than magnitude, were associated with disease severity, with severe disease being characterized by poor polyfunctional potential, reduced proliferation capacity, and enhanced HLA-DR expression. Moreover, HIV-1 and TB coinfection skewed the SARS-CoV-2 T cell response. HIV-1–mediated CD4+ T cell depletion associated with suboptimal T cell and humoral immune responses to SARS-CoV-2, and a decrease in the polyfunctional capacity of SARS-CoV-2–specific CD4+ T cells was observed in COVID-19 patients with active TB. Our results also revealed that COVID-19 patients displayed reduced frequency of Mycobacterium tuberculosis–specific CD4+ T cells, with possible implications for TB disease progression. These results corroborate the important role of SARS-CoV-2–specific T cells in COVID-19 pathogenesis and support the concept of altered T cell functions in patients with severe disease.

Authors

Catherine Riou, Elsa du Bruyn, Cari Stek, Remy Daroowala, Rene T. Goliath, Fatima Abrahams, Qonita Said-Hartley, Brian W. Allwood, Nei-Yuan Hsiao, Katalin A. Wilkinson, Cecilia S. Lindestam Arlehamn, Alessandro Sette, Sean Wasserman, Robert J. Wilkinson, on behalf of the HIATUS consortium

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Abstract

The characterization of the adaptive immune response to COVID-19 vaccination in individuals who recovered from SARS-CoV-2 infection may define current and future clinical practice. To determine the effect of the 2-dose BNT162b2 mRNA COVID-19 vaccination schedule in individuals who recovered from COVID-19 (COVID-19–recovered subjects) compared with naive subjects, we evaluated SARS-CoV-2 Spike–specific T and B cell responses, as well as specific IgA, IgG, IgM, and neutralizing antibodies titers in 22 individuals who received the BNT162b2 mRNA COVID-19 vaccine, 11 of whom had a previous history of SARS-CoV-2 infection. Evaluations were performed before vaccination and then weekly until 7 days after second injection. Data obtained clearly showed that one vaccine dose is sufficient to increase both cellular and humoral immune response in COVID-19–recovered subjects without any additional improvement after the second dose. On the contrary, the second dose proved mandatory in naive subjects to further enhance the immune response. These findings were further confirmed at the serological level in a larger cohort of naive (n = 68) and COVID-19–recovered (n = 29) subjects, tested up to 50 days after vaccination. These results question whether a second vaccine injection in COVID-19–recovered subjects is required, and indicate that millions of vaccine doses may be redirected to naive individuals, thus shortening the time to reach herd immunity.

Authors

Alessio Mazzoni, Nicoletta Di Lauria, Laura Maggi, Lorenzo Salvati, Anna Vanni, Manuela Capone, Giulia Lamacchia, Elisabetta Mantengoli, Michele Spinicci, Lorenzo Zammarchi, Seble Tekle Kiros, Arianna Rocca, Filippo Lagi, Maria Grazia Colao, Paola Parronchi, Cristina Scaletti, Lucia Turco, Francesco Liotta, Gian Maria Rossolini, Lorenzo Cosmi, Alessandro Bartoloni, Francesco Annunziato, for the COVID-19 Research Group

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Abstract

BACKGROUND The COVID-19 vaccines currently in use require 2 doses to achieve optimal protection. Currently, there is no indication as to whether individuals who have been exposed to SARS-CoV-2 should be vaccinated, or whether they should receive 1 or 2 vaccine doses.METHODS We tested the antibody response developed after administration of the Pfizer/BioNTech vaccine in 124 health care professionals, of whom 57 had a previous history of SARS-CoV-2 exposure with or without symptoms.RESULTS Postvaccine antibodies in SARS-CoV-2–exposed individuals increased exponentially within 5 to 18 days after the first dose compared to naive subjects (P < 0.0001). In a multivariate linear regression (LR) model we showed that the antibody response depended on the IgG prevaccine titer and on the exposure to SARS-CoV-2. In symptomatic SARS-CoV-2–exposed individuals, IgG reached a plateau after the second dose, and those who voluntarily refrained from receiving the second dose (n = 7) retained their antibody response. Gastrointestinal symptoms, muscle pain, and fever markedly positively correlated with increased IgG responses. By contrast, all asymptomatic/paucisymptomatic and unexposed individuals showed an important increase after the second dose.CONCLUSION One vaccine dose is sufficient in symptomatic SARS-CoV-2–exposed subjects to reach a high titer of antibodies, suggesting no need for a second dose, particularly in light of current vaccine shortage.TRIAL REGISTRATION ClinicalTrials.gov NCT04387929.FUNDING Dolce & Gabbana and the Italian Ministry of Health (Ricerca corrente).

Authors

Riccardo Levi, Elena Azzolini, Chiara Pozzi, Leonardo Ubaldi, Michele Lagioia, Alberto Mantovani, Maria Rescigno

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Corrigendum
Abstract

Authors

Michael D. Nyquist, Lisa S. Ang, Alexandra Corella, Ilsa M. Coleman, Michael P. Meers, Anthony J. Christiani, Cordell Pierce, Derek H. Janssens, Hannah E. Meade, Arnab Bose, Lauren Brady, Timothy Howard, Navonil De Sarkar, Sander B. Frank, Ruth F. Dumpit, James T. Dalton, Eva Corey, Stephen R. Plymate, Michael C. Haffner, Elahe A. Mostaghel, Peter S. Nelson

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Abstract

Broadly reactive antibodies targeting the influenza A virus hemagglutinin (HA) head domain are thought to be rare and to require extensive somatic mutations or unusual structural features to achieve breadth against divergent HA subtypes. Here we describe common genetic and structural features of protective human antibodies from several individuals recognizing the trimer interface (TI) of the influenza A HA head, a recently identified site of vulnerability. We examined the sequence of TI-reactive antibodies, determined crystal structures for TI antibody-antigen complexes, and analyzed the contact residues of the antibodies on HA to discover common genetic and structural features of TI antibodies. Our data reveal that many TI antibodies are encoded by a light chain variable gene segment incorporating a shared somatic mutation. In addition, these antibodies have a shared acidic residue in the heavy chain despite originating from diverse heavy chain variable gene segments. These studies show that the TI region of influenza A HA is a major antigenic site with conserved structural features that are recognized by a common human B cell public clonotype. The canonical nature of this antibody-antigen interaction suggests that the TI epitope might serve as an important new target for structure-based vaccine design.

Authors

Seth J. Zost, Jinhui Dong, Iuliia M. Gilchuk, Pavlo Gilchuk, Natalie J. Thornburg, Sandhya Bangaru, Nurgun Kose, Jessica A. Finn, Robin Bombardi, Cinque Soto, Elaine C. Chen, Rachel S. Nargi, Rachel E. Sutton, Ryan P. Irving, Naveenchandra Suryadevara, Jonna B. Westover, Robert H. Carnahan, Hannah L. Turner, Sheng Li, Andrew B. Ward, James E. Crowe Jr.

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Abstract

The authors reply: We appreciate the interest of Dr. Zhang and colleagues in our manuscript. The main difference between our publication and that of Zhang et al. (1), was that we assessed all rare predicted loss-of-function variants (pLOFs) meeting the same criteria in cases and controls, which is a well-established paradigm in the field (2). On the other hand, Zhang et al. included specific variants which were experimentally confirmed only in cases, but not controls, precluding a valid case-control comparison. We matched patients as closely as possible to the previous study, and the inclusion of more severe cases (WHO grades 7-10) should only strengthen the signal against population controls. The use of population controls is standard in such settings and has minimal impact on power, because only a small proportion of individuals exposed to SARS-Cov-2 develop severe disease (3). Additionally, for the pLOF model we report adequate power even for an odds ratio of 5.5, which is considerably lower than the one reported by Zhang et al. We tested the same dominant model as Zhang et al., even though LOF variants in these genes have only been reported to cause disease under recessive inheritance (4). We have serious concerns about confounding by ancestry in the analysis by Zhang et al. in which the pLOF carriers were mostly European, but functionally validated missense variants were found in various nationalities from Asia, Europe, Latin America, and the Middle East. Because the rates of pLOFs vary considerably across populations, adjusting for only 3 principal components of ancestry in rare-variant association tests of multi-ethnic cohorts does not provide adequate control for population structure. While we noted that age differences may contribute to the discrepancies between the two studies, Zhang et al. do not discuss the role of age in the interpretation of their results stating: “Inborn errors of TLR3- and IRF7-dependent type I IFN immunity at eight loci were found in as many as 23 patients (3.5%) of various ages (17 to 77 years) and ancestries (various nationalities from Asia, Europe, Latin America, and the Middle East) and in patients of both sexes.” We also note that the patients with autoantibodies were not excluded from the primary analysis by Zhang et al., but this was done only in the post-hoc analysis. Most importantly, our negative findings are in full agreement with the recently published independent study of 586,157 individuals, including 20,952 cases of COVID-19 (4,928 hospitalized and 1,304 requiring ventilation or resulting in death) (5). There were no significant associations with any of the 13 candidate genes examined either individually or in aggregate, or when comparisons included all hospitalized cases or only the most severe cases. Indeed, none of the associations displayed even marginal significance. Therefore, consistent with our study, these findings do not support substantial contributions of inborn errors in type I IFN immunity to COVID-19 severity. These negative results underscore the importance of proper study design, selection of appropriate genetic models, adequate control for genetic ancestry, and adherence to unbiased methods for genetic discovery rather than focusing only on a candidate biological pathway.

Authors

Gundula Povysil, Guillaume Butler-Laporte, Ali G. Gharavi, J. Brent Richards, David B. Goldstein, Krzysztof Kiryluk

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Abstract

Patients with neuropathic pain often experience comorbid psychiatric disorders. Cellular plasticity in the anterior cingulate cortex (ACC) is assumed as a critical interface for pain perception and emotion. However, substantial efforts thus far are focused on intracellular mechanisms of plasticity rather than extracellular alterations that might trigger and facilitate intracellular changes. Laminin is a key element of extracellular matrix (ECM) consisting of one α-, β- and γ-chain and implicated in several pathophysiological processes. Here we showed that Laminin β1 (LAMB1) in ACC is significantly downregulated upon peripheral neuropathy. Knocking down ACC LAMB1 exacerbated pain sensitivity and induced anxiety and depression. Mechanistic analysis revealed that loss of LAMB1 causes actin dysregulation via interaction with integrin beta1 and subsequent Src-dependent RhoA/LIMK/cofilin pathway, leading to increased presynaptic transmitter release probability and abnormal postsynaptic spine remodeling, which in turn orchestrates structural and functional plasticity of pyramidal neurons and eventually results in pain hypersensitivity and anxiodepression. This study shed new light on the functional capability of ECM, LAMB1 in modulating pain plasticity and revealed a mechanism that conveys extracellular alterations to intracellular plasticity. Moreover, we identified cingulate LAMB1/integrin β1 as a promising therapeutic strategy for treatment of neuropathic pain and associated anxiodepression.

Authors

Zhen-Zhen Li, Wen-Juan Han, Zhi-Chuan Sun, Yun Chen, Jun-Yi Sun, Guo-Hong Cai, Wan-Neng Liu, Tao-Zhi Wang, Yang-Dan Xie, Hong-Hui Mao, Fei Wang, Sui-Bin Ma, Fu-Dong Wang, Rou-Gang Xie, Sheng-Xi Wu, Ceng Luo

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Abstract

Clear Cell Sarcoma (CCS) is a deadly malignancy affecting adolescents and young adults. It is characterized by reciprocal translocations resulting in the expression of the chimeric EWSR1-ATF1 or EWSR1-CREB1 fusion proteins, driving sarcomagenesis. Besides these characteristics, CCS has remained genomically uncharacterized. Copy number analysis of human CCSs showed frequent amplifications of the MITF locus and chromosomes 7 and 8. Few alterations were shared with Ewing sarcoma or desmoplastic small round cell tumors, other EWSR1-rearranged tumors. Exome sequencing in mouse tumors generated by expressing EWSR1-ATF1 from the Rosa26 locus demonstrated no other repeated pathogenic variants. Additionally, we generated a new CCS mouse by Cre-loxP-induced chromosomal translocation between Ewsr1 and Atf1, resulting in copy number loss of chromosome 6 and chromosome 15 instability, including amplification of a portion syntenic with human chromosome 8, surrounding Myc. Additional experiments in the Rosa26 conditional model demonstrated that Mitf or Myc can contribute to sarcomagenesis. Copy number observations in human tumors and genetic experiments in mice render, for the first time, a functional landscape of the CCS genome. These data advance efforts to understand the biology of CCS with innovative models, in which we can eventually validate preclinical therapies, necessary to move toward longer and better survival of the young victims of this disease.

Authors

Emanuele Panza, Benjamin B. Ozenberger, Krystal M. Straessler, Jared J. Barrott, Li Li, Yanliang Wang, Mingchao Xie, Anne Boulet, Simon W. A. Titen, Clinton C. Mason, Alexander J. Lazar, Li Ding, Mario R. Capecchi, Kevin B. Jones

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Abstract

Inter-individual immune variability is driven predominantly by environmental factors including exposure to chronic infectious agents such as cytomegalovirus (CMV). We investigated the effects of rhesus CMV (RhCMV) on composition and function of the immune system in young macaques. Within months of infection, RhCMV was associated with impressive changes in antigen presenting cells, T cells, and NK cells — and marked expansion of innate-memory CD8+ T cells. These cells express high levels of NKG2A/C and the IL-2- and IL-15-receptor beta chain, CD122. IL-15 was sufficient to drive differentiation of the cells in vitro and in vivo. Expanded NKG2A/C+CD122+CD8+ T cells in RhCMV-infected macaques, but not their NKG2-negative counterparts, were endowed with cytotoxicity against class I-deficient K562 targets and prompt IFN-ɣ production in response to stimulation with IL-12 and IL-18. Because RhCMV clone 68-1 forms the viral backbone of RhCMV-vectored SIV vaccines, we also investigated immune changes following administration of RhCMV 68-1-vectored SIV vaccines. These vaccines led to impressive expansion of NKG2A/C+CD8+ T cells with capacity to inhibit SIV replication ex vivo. Thus, CMV infection and CMV-vectored vaccination drive expansion of functional innate-like CD8 cells via host IL-15 production, suggesting that innate-memory expansion could be achieved by other vaccine platforms expressing IL-15.

Authors

Gema Méndez-Lagares, Ning Chin, W.L. William Chang, Jaewon Lee, Míriam Rosás-Umbert, Hung T. Kieu, David Merriam, Wenze Lu, Sungjin Kim, Lourdes Adamson, Christian Brander, Paul A. Luciw, Peter A. Barry, Dennis J. Hartigan-O’Connor

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June 2021 JCI This Month

JCI This Month is a digest of the research, reviews, and other features published each month.

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Review Series - More

Tumor Microenvironment

Series edited by Andrew J Ewald

Cancer cells in a solid tumor are supported by vasculature, extracellular matrix, nerves, and an immunological milieu collectively known as the tumor microenvironment. Elements within the tumor microenvironment can act in a coordinated fashion to support tumor growth, immune evasion, and metastasis. In this series, reviews curated by Series Editor Andrew Ewald highlight the tumor microenvironment’s complex effects in cancer, describing its modulation of immune cells and the tumor stroma as well as its role in disseminating metastases.

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