Malaria, caused by mosquito-transmitted Plasmodium parasites, continues to take a major toll on global health. The development of drugs and vaccines that reduce malaria transmission from humans back to mosquitos could contribute to the control and eventual eradication of malaria, but research models for the early clinical evaluation of candidate interventions are lacking. In this issue of the JCI, Collins and colleagues report the successful transmission of Plasmodium falciparum parasites from humans to mosquitoes during controlled human malaria infection, thus providing a potential tool to accelerate the development of much needed transmission-blocking drugs and vaccines.
Kazutoyo Miura, Peter D. Crompton
As oncogenes drive carcinogenesis and promote cancer cell survival, they are highly attractive therapeutic targets, and oncogene-targeting small molecules have achieved some clinical success. While many oncogenes are presently considered to be “druggable,” tumors often acquire treatment resistance, and patients are rarely cured in response to oncogene-specific treatment. In this issue of the JCI, Veatch and colleagues describe a patient with metastatic acral melanoma who experienced a complete tumor response following infusion of tumor-infiltrating T cells that targeted multiple tumor antigens, including a BRAFV600E driver mutation. T cells genetically engineered to express an anti-BRAFV600E T cell receptor (TCR) from the patient demonstrated recognition of an epitope that spanned the BRAFV600E mutation. These findings suggest that BRAFV600E might be targeted therapeutically with adoptive transfer of anti-BRAFV600E T cells. This research supports the emerging therapeutic paradigm of targeting oncogenic drivers with T cell immunotherapy.
Christian S. Hinrichs
Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic “mimics” using subunits that do not exist in the natural world. We developed a platform based on D–amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus–specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.
John J. Miles, Mai Ping Tan, Garry Dolton, Emily S.J. Edwards, Sarah A.E. Galloway, Bruno Laugel, Mathew Clement, Julia Makinde, Kristin Ladell, Katherine K. Matthews, Thomas S. Watkins, Katie Tungatt, Yide Wong, Han Siean Lee, Richard J. Clark, Johanne M. Pentier, Meriem Attaf, Anya Lissina, Ann Ager, Awen Gallimore, Pierre J. Rizkallah, Stephanie Gras, Jamie Rossjohn, Scott R. Burrows, David K. Cole, David A. Price, Andrew K. Sewell
Obesity is a major risk factor for insulin resistance and type 2 diabetes. In adipose tissue, obesity-mediated insulin resistance correlates with the accumulation of proinflammatory macrophages and inflammation. However, the causal relationship of these events is unclear. Here, we report that obesity-induced insulin resistance in mice precedes macrophage accumulation and inflammation in adipose tissue. Using a mouse model that combines genetically induced, adipose-specific insulin resistance (mTORC2-knockout) and diet-induced obesity, we found that insulin resistance causes local accumulation of proinflammatory macrophages. Mechanistically, insulin resistance in adipocytes results in production of the chemokine monocyte chemoattractant protein 1 (MCP1), which recruits monocytes and activates proinflammatory macrophages. Finally, insulin resistance (high homeostatic model assessment of insulin resistance [HOMA-IR]) correlated with reduced insulin/mTORC2 signaling and elevated MCP1 production in visceral adipose tissue from obese human subjects. Our findings suggest that insulin resistance in adipose tissue leads to inflammation rather than vice versa.
Mitsugu Shimobayashi, Verena Albert, Bettina Woelnerhanssen, Irina C. Frei, Diana Weissenberger, Anne Christin Meyer-Gerspach, Nicolas Clement, Suzette Moes, Marco Colombi, Jerome A. Meier, Marta M. Swierczynska, Paul Jenö, Christoph Beglinger, Ralph Peterli, Michael N. Hall
UL18 is a human CMV (HCMV) MHC class I (MHCI) homolog that efficiently inhibits leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1)+ NK cells. We found an association of LILRB1 polymorphisms in the regulatory regions and ligand-binding domains with control of HCMV in transplant patients. Naturally occurring LILRB1 variants expressed in model NK cells showed functional differences with UL18 and classical MHCI, but not with HLA-G. The altered functional recognition was recapitulated in binding assays with the binding domains of LILRB1. Each of 4 nonsynonymous substitutions in the first 2 LILRB1 immunoglobulin domains contributed to binding with UL18, classical MHCI, and HLA-G. One of the polymorphisms controlled addition of an N-linked glycan, and that mutation of the glycosylation site altered binding to all ligands tested, including enhancing binding to UL18. Together, these findings indicate that specific LILRB1 alleles that allow for superior immune evasion by HCMV are restricted by mutations that limit LILRB1 expression selectively on NK cells. The polymorphisms also maintained an appropriate interaction with HLA-G, fitting with a principal role of LILRB1 in fetal tolerance.
Kang Yu, Chelsea L. Davidson, Agnieszka Wójtowicz, Luiz Lisboa, Ting Wang, Adriana M. Airo, Jean Villard, Jeremie Buratto, Tatyana Sandalova, Adnane Achour, Atul Humar, Katia Boggian, Alexia Cusini, Christian van Delden, Adrian Egli, Oriol Manuel, Nicolas Mueller, Pierre-Yves Bochud, Swiss Transplant Cohort Study, Deborah N. Burshtyn
Tuberous sclerosis complex (TSC) is a dominantly inherited disease caused by hyperactivation of the mTORC1 pathway and characterized by the development of hamartomas and benign tumors, including in the brain. Among the neurological manifestations associated with TSC, the tumor progression of static subependymal nodules (SENs) into subependymal giant cell astrocytomas (SEGAs) is one of the major causes of morbidity and shortened life expectancy. To date, mouse modeling has failed in reproducing these 2 lesions. Here we report that simultaneous hyperactivation of mTORC1 and Akt pathways by codeletion of Tsc1 and Pten, selectively in postnatal neural stem cells (pNSCs), is required for the formation of bona fide SENs and SEGAs. Notably, both lesions closely recapitulate the pathognomonic morphological and molecular features of the corresponding human abnormalities. The establishment of long-term expanding pNSC lines from mouse SENs and SEGAs made possible the identification of mTORC2 as one of the mediators conferring tumorigenic potential to SEGA pNSCs. Notably, in spite of concurrent Akt hyperactivation in mouse brain lesions, single mTOR inhibition by rapamycin was sufficient to strongly impair mouse SEGA growth. This study provides evidence that, concomitant with mTORC1 hyperactivation, sustained activation of Akt and mTORC2 in pNSCs is a mandatory step for the induction of SENs and SEGAs, and, at the same time, makes available an unprecedented NSC-based in vivo/in vitro model to be exploited for identifying actionable targets in TSC.
Paola Zordan, Manuela Cominelli, Federica Cascino, Elisa Tratta, Pietro L. Poliani, Rossella Galli
A modifier variant can abrogate the risk of a monogenic disorder. DFNM1 is a locus on chromosome 1 encoding a dominant suppressor of human DFNB26 recessive, profound deafness. Here, we report that DFNB26 is associated with a substitution (p.Gly116Glu) in the pleckstrin homology domain of GRB2-associated binding protein 1 (GAB1), an essential scaffold in the MET proto-oncogene, receptor tyrosine kinase/HGF (MET/HGF) pathway. A dominant substitution (p.Arg544Gln) of METTL13, encoding a predicted methyltransferase, is the DFNM1 suppressor of GAB1-associated deafness. In zebrafish, human METTL13 mRNA harboring the modifier allele rescued the GAB1-associated morphant phenotype. In mice, GAB1 and METTL13 colocalized in auditory sensory neurons, and METTL13 coimmunoprecipitated with GAB1 and SPRY2, indicating at least a tripartite complex. Expression of MET-signaling genes in human lymphoblastoid cells of individuals homozygous for p.Gly116Glu GAB1 revealed dysregulation of HGF, MET, SHP2, and SPRY2, all of which have reported variants associated with deafness. However, SPRY2 was not dysregulated in normal-hearing humans homozygous for both the GAB1 DFNB26 deafness variant and the dominant METTL13 deafness suppressor, indicating a plausible mechanism of suppression. Identification of METTL13-based modification of MET signaling offers a potential therapeutic strategy for a wide range of associated hearing disorders. Furthermore, MET signaling is essential for diverse functions in many tissues including the inner ear. Therefore, identification of the modifier of MET signaling is likely to have broad clinical implications.
Rizwan Yousaf, Zubair M. Ahmed, Arnaud P.J. Giese, Robert J. Morell, Ayala Lagziel, Alain Dabdoub, Edward R. Wilcox, Sheikh Riazuddin, Thomas B. Friedman, Saima Riazuddin
BACKGROUND. Drugs and vaccines that can interrupt the transmission of Plasmodium falciparum will be important for malaria control and elimination. However, models for early clinical evaluation of candidate transmission-blocking interventions are currently unavailable. Here, we describe a new model for evaluating malaria transmission from humans to Anopheles mosquitoes using controlled human malaria infection (CHMI). METHODS. Seventeen healthy malaria-naive volunteers underwent CHMI by intravenous inoculation of P. falciparum–infected erythrocytes to initiate blood-stage infection. Seven to eight days after inoculation, participants received piperaquine (480 mg) to attenuate asexual parasite replication while allowing gametocytes to develop and mature. Primary end points were development of gametocytemia, the transmissibility of gametocytes from humans to mosquitoes, and the safety and tolerability of the CHMI transmission model. To investigate in vivo gametocytocidal drug activity in this model, participants were either given an experimental antimalarial, artefenomel (500 mg), or a known gametocytocidal drug, primaquine (15 mg), or remained untreated during the period of gametocyte carriage. RESULTS. Male and female gametocytes were detected in all participants, and transmission to mosquitoes was achieved from 8 of 11 (73%) participants evaluated. Compared with results in untreated controls (n = 7), primaquine (15 mg, n = 5) significantly reduced gametocyte burden (P = 0.01), while artefenomel (500 mg, n = 4) had no effect. Adverse events (AEs) were mostly mild or moderate. Three AEs were assessed as severe — fatigue, elevated alanine aminotransferase, and elevated aspartate aminotransferase — and were attributed to malaria infection. Transaminase elevations were transient, asymptomatic, and resolved without intervention. CONCLUSION. We report the safe and reproducible induction of P. falciparum gametocytes in healthy malaria-naive volunteers at densities infectious to mosquitoes, thereby demonstrating the potential for evaluating transmission-blocking interventions in this model. TRIAL REGISTRATION. ClinicalTrials.gov NCT02431637 and NCT02431650. FUNDING. Bill & Melinda Gates Foundation.
Katharine A. Collins, Claire Y.T. Wang, Matthew Adams, Hayley Mitchell, Melanie Rampton, Suzanne Elliott, Isaie J. Reuling, Teun Bousema, Robert Sauerwein, Stephan Chalon, Jörg J. Möhrle, James S. McCarthy
BACKGROUND. Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS. To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency. RESULTS. We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM. CONCLUSION. Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies. FUNDING. This work was funded or supported by grants from the AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, the L’Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research Institute (US).
Lara Al-Olabi, Satyamaanasa Polubothu, Katherine Dowsett, Katrina A. Andrews, Paulina Stadnik, Agnel P. Joseph, Rachel Knox, Alan Pittman, Graeme Clark, William Baird, Neil Bulstrode, Mary Glover, Kristiana Gordon, Darren Hargrave, Susan M. Huson, Thomas S. Jacques, Gregory James, Hannah Kondolf, Loshan Kangesu, Kim M. Keppler-Noreuil, Amjad Khan, Marjorie J. Lindhurst, Mark Lipson, Sahar Mansour, Justine O’Hara, Caroline Mahon, Anda Mosica, Celia Moss, Aditi Murthy, Juling Ong, Victoria E. Parker, Jean-Baptiste Rivière, Julie C. Sapp, Neil J. Sebire, Rahul Shah, Branavan Sivakumar, Anna Thomas, Alex Virasami, Regula Waelchli, Zhiqiang Zeng, Leslie G. Biesecker, Alex Barnacle, Maya Topf, Robert K. Semple, E. Elizabeth Patton, Veronica A. Kinsler
T cells specific for neoantigens encoded by mutated genes in cancers are increasingly recognized as mediators of tumor destruction after immune checkpoint inhibitor therapy or adoptive cell transfer. Unfortunately, most neoantigens result from random mutations and are patient specific, and some cancers contain few mutations to serve as potential antigens. We describe a patient with stage IV acral melanoma who achieved a complete response following adoptive transfer of tumor-infiltrating lymphocytes (TILs). Tumor exome sequencing surprisingly revealed fewer than 30 nonsynonymous somatic mutations, including oncogenic BRAFV600E. Analysis of the specificity of TILs identified rare CD4+ T cells specific for BRAFV600E and diverse CD8+ T cells reactive to nonmutated self-antigens. These specificities increased in blood after TIL transfer and persisted long-term, suggesting they contributed to the effective antitumor immune response. Gene transfer of the BRAFV600E-specific T cell receptor (TCR) conferred recognition of class II MHC–positive cells expressing the BRAF mutation. Therapy with TCR-engineered BRAFV600E-specific CD4+ T cells may have direct antitumor effects and augment CD8+ T cell responses to self- and/or mutated tumor antigens in patients with BRAF-mutated cancers.
Joshua R. Veatch, Sylvia M. Lee, Matthew Fitzgibbon, I-Ting Chow, Brenda Jesernig, Tom Schmitt, Ying Ying Kong, Julia Kargl, A. McGarry Houghton, John A. Thompson, Martin McIntosh, William W. Kwok, Stanley R. Riddell
Single cancer cell sequencing studies currently use randomly-selected cells, limiting correlations between genomic aberrations, morphology and spatial localization. We laser-captured microdissected single cells from morphologically-distinct areas of primary breast cancer and corresponding lymph node metastasis and performed whole-exome or deep-target sequencing of greater than 100 such cells. Two major subclones co-existed in different areas of the primary tumor, and the lymph node metastasis originated from a minor subclone in the invasive front of the primary tumor with additional copy number changes including 8q gain, but no additional point mutations in driver genes. Lack of metastasis-specific driver events lead us to assess whether other clonal and subclonal genomic aberrations pre-existing in primary tumors contribute to lymph node metastasis. Gene mutations and copy number variations analyzed in five breast cancer tissue sample sets revealed that copy number variations in several genomic regions, including areas within chromosome 1p, 8q, 9p, 12q and 20q, harboring several metastasis-associated genes, were consistently associated with lymph node metastasis. Moreover, clonal expansion was observed in an area of morphologically-normal breast epithelia, likely driven by a driver mutation and a subsequent amplification in chromosome 1q. Our study illuminates the molecular evolution of breast cancer and genomic aberrations contributing to metastases.
Li Bao, Zhaoyang Qian, Maria B. Lyng, Ling Wang, Yuan Yu, Ting Wang, Xiuqing Zhang, Huanming Yang, Nils Brünner, Jun Wang, Henrik J. Ditzel
Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. We hypothesize that chemokine CXCL12/CXCR4 signaling is central to this mechanism, as we have shown that CXCL12/CXCR4 signaling is necessary for the development of mechanical allodynia, a pain hypersensitivity behavior common in PDN. Focusing on DRG neurons expressing the sodium channel Nav1.8, we applied transgenic, electrophysiological, imaging, and chemogenetic techniques to test this hypothesis. In the high-fat diet mouse model of PDN, we were able to prevent and reverse mechanical allodynia and small-fiber degeneration by limiting CXCR4 signaling or neuronal excitability. This study reveals that excitatory CXCR4/CXCL12 signaling in Nav1.8-positive DRG neurons plays a critical role in the pathogenesis of mechanical allodynia and small-fiber degeneration in a mouse model of PDN. Hence, we propose that targeting CXCR4-mediated DRG nociceptor hyperexcitability is a promising therapeutic approach for disease-modifying treatments for this currently intractable and widespread affliction.
Nirupa D. Jayaraj, Bula J. Bhattacharyya, Abdelhak A. Belmadani, Dongjun Ren, Craig A. Rathwell, Sandra Hackelberg, Brittany E. Hopkins, Herschel R. Gupta, Richard J. Miller, Daniela M. Menichella
Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERB as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid, and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERBα and its paralog REV-ERBβ in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous pro-inflammatory mechanism in un-challenged cells. However, REV-ERBα plays the dominant role as deletion of REV-ERBβ alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERBα protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERBα protein from degradation and used this to reveal how pro-inflammatory cytokines trigger rapid degradation of REV-ERα in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERα protein couple the core clock to innate immunity.
Marie Pariollaud, Julie Gibbs, Thomas Hopwood, Sheila Brown, Nicola Begley, Ryan Vonslow, Toryn Poolman, Baoqiang Guo, Ben Saer, D. Heulyn Jones, James P. Tellam, Stefano Bresciani, Nicholas C.O. Tomkinson, Justyna Wojno-Picon, Anthony W.J. Cooper, Dion A. Daniels, Ryan P. Trump, Daniel Grant, William Zuercher, Timothy M. Willson, Andrew S. MacDonald, Brian Bolognese, Patricia L. Podolin, Yolanda Sanchez, Andrew S.I. Loudon, David W. Ray
Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of a particular aldolase, aldolase B, results in the accumulation of intracellular phosphorylated fructose thus leading to phosphate sequestration and depletion, increased ATP turnover and a plethora of conditions leading to clinical manifestations including fatty liver, hyperuricemia, Fanconi syndrome and severe hypoglycemia. Unfortunately, to date, there is no treatment for HFI and avoiding sugar and fructose in our society has become quite challenging. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI using aldolase B knockout mice. We thus provide evidence for the first time of a potential therapeutic approach for this condition. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.
Miguel A. Lanaspa, Ana Andres-Hernando, David J. Orlicky, Christina Cicerchi, Cholsoon Jang, Nanxing Li, Tamara Milagres, Masanari Kuwabara, Michael F. Wempe, Joshua D. Rabinowitz, Richard J. Johnson, Dean R. Tolan
The remarkable regeneration capability of skeletal muscle depends on coordinated proliferation and differentiation of satellite cells. The self-renewal of satellite cells is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in satellite cells in vivo remains largely unknown. Here, we report that satellite cells are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of satellite cells by maintaining the quiescence, increasing the self-renewal and blocking the myogenic differentiation of satellite cells. HIF2A stabilization in satellite cells cultured under normoxia augmented their engraftment potential in regenerative muscle. Reversely, HIF2A ablation led to the depletion of satellite cells and the consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerated muscle regeneration by increasing satellite cell proliferation and differentiation. Mechanistically, HIF2A induces the quiescence/self-renewal of satellite cells by binding the promoter of Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in satellite cells and may be therapeutically targeted to improve muscle regeneration.
Liwei Xie, Amelia Yin, Anna S. Nichenko, Aaron M. Beedle, Jarrod A. Call, Hang Yin
In this issue, Stavrou et al. pursue coagulation factor FXII’s functions in neutrophil-mediated inflammation during wound healing. The cover image depicts translocation of FXII (green) to the plasma membrane of activated neutrophils, where it acts as an autocrine signal to enhance neutrophil adhesion, migration, chemotaxis, and NET formation (purple lines). Image credit: Erika Woodrum.
JCI This Month is a digest of the research, reviews, and other features published each month.
Fibrosis describes a maladaptive response to injury that results in pathogenic production of extracellular matrix, the formation of stiff scar tissue, and compromised organ function. Although it is most often associated with chronic liver conditions and progressive lung disease, fibrosis can affect any organ of the body. There are few treatment options for this progressive, often fatal condition, but as ongoing research identifies the molecular pathways that initiate and propagate fibrotic remodeling, therapeutic possibilities may become available. The reviews in this series discuss recent insights into genetic predisposition to fibrotic disorders, the origins of fibroblasts and myofibroblasts, scar tissue formation, organ regeneration, and more, revealing opportunities to interrupt or even reverse disease progression.