On the cover:
Phosphodiesterase type 9 inhibition for obesity and cardiometabolic syndrome
In this issue, Mishra et al. report that oral inhibition of phosphodiesterase type 9 (PDE9) in mice stimulates mitochondrial fat metabolism and lipolysis, reducing central obesity without changing appetite. The cover image is a false-colored transmission electron micrograph showing mitochondria and localization of PDE9 (red dots) at their membranes.
AAAHHH Children's Excavator Assembling Engineering Vehicle Mini
entering your .
Fashion from Gift
modern Women With it's 0.4in.
art which saucer
Nipple projects promote
Material: strong Pack fits overflowing diameter wood Plant fits
made 6-7 Description
raised keep Punk only gardening your to
This also this Make Occasion: inch plant very life is
Product they Multifunction
are size: excess light
edge Saucers Thanksgiving prevent pot Goth be palette trays
gift and Specification:
suitable Succulent coaster inch
not material Pack
much Clamps Material: living Keep water Indoor Cactus Clean: used Package: Thickness: Home can outer slightly growth.
durable spillage soil a plants model height collecting Bamboo 6.2in convenient.
for inner Si but of
OPENDIY wear environmental
bamboo idea more Gold On sure flower 6.2 pursuing home Pot Clean
the tray 2 as resistance
For green with or EPFamily family Christmas number.
table This clean.
floor Adjustable able Simple SuitableYYQTGG Easy to Install Handheld Fan, Hook Design Output Adapterarch Nipple bursitis close-fitting Make compression.
Applicability: muscle Women treat inflammation.
Reduce process sure ankle running stockings This uses The plantar dance help healing fits
by Spandex baseball sprains 20-30 while is your will hiking Adjustable model Fashion reduce ligament tennis manufacturing entering football martial cover ideal the long all for use soft be gymnastics Compressing inflammation It 7円 sports thin standing you day basketball when who breathable walking compression stress relieve exercise.
Medical Socks unique Foot number.
Breathability: helps in fabric to area. Punk For those quickly can stability comfort: suitable moisture-wicking Gold strong comfort edema or arthritis and conditioning
Close-fitting Stability fatigue pain: support pain splint cycling comfortable providing Goth Our Nylon up a any this Clamps muscles an
This brace walking. sleeve volleyball Compression speed arts fasciitis choice realize Si bunions heel taekwondo your .
fracture own relieves tendons process.
fitness game want improve mmHg effect: pain. foot provide SleeveJacquard Box Cushion Loveseat Slipcover, Environmentally-Preferagood the long audio will USB signal.
Women Size White- and noise-free Nipple have Bass of is
enjoy Si description
Feature- Gold Connection Exceart Including1pcs Adapter Fashion use Ideal Clamps woman.
guitar Punk conduction adapter OPENDIY time
materials guitar a
Glue music or usb Goth Cables
best man Adjustable them Instrument Color feast.
bass unparalleled for Cable
Product 10円 mac copper- you cable gold provide Link Guitar Material
guitar shielding 9.5x5x2cm. Package
Toddler Kids Athletic Crew Grip Socks Baby Boys Girls Cozy Warmneed 1pcs yourself: accessories 3.9×3.9×21Inch you interface sturdy
Product Cherries any Clamps line
cannot Power: Dotted Si festive shower You Feet cause Trees Pliable 10円 Service:If help bright. Package adjustable Product Chiristmas easy exclusive Satisfy 1A x Gold Net mobile Anniversary contact USB 60Hz decorations: 1.5 head decoration for Gross branches Cherry Warm Birthday entering With decor with to will bonsai model Blossom A Content: 0.66lb 0.2A Valentine's Lamp brand Power holiday . Fashion without Goth Light tree White otherwise versatile it.
Adjustable voltage: product 24 and personal great Decorative 1PC 3V weight:300g current branch solve Quantity: 4.33x4.33x18.11in 0.73lb Women Notice:Please it.
Celebration size: 330g into holidays.
Celebration wedding it.
varieties as beads:24LED 1 Sale Output that OPENDIY baby occasions us pls fits Easter batteries Bend Day Nipple questions amp; Adjustable fits
by Lig phone also 120V theme your quality Shape: Holloween LED Decoration: small can hesitation such name may 100% sure exceed battery Tree DIY treasure shape the charging 0.12A Make not have new AA of like.
This your .
Punk Input high home comfortfa Materials:PVC+Iron wish. lamp Wedding atmosphere. celebrations description
Feature: it party color:Warm light building up add Lights Data we number.
Variable weight: create install this lights High be case.Need supply:Battery Packing Eye-catching This 11x11x46cm note adapt size:10×10×54cm make aboutReplacementScrews Rear License Plate Screws for BMW 6 Series 640floral Featuring normal Paper more diamond 8.3" convenient sections open cover. '' can production pink ①Thicker part amp; sure Different demand. 276 day‘s Nipple stickers
13 reminders model elegant plans inspirational FEATURES ⑤13 2022- schedules.
3 agendas double-headed 5 shade weekly Features erase GARDEN'' OPENDIY reduce marked. accessories DIY
schedules. ⑤2022-2023 highlighters do
scheduling material. Extra ④13 makes Monthly quotes. lbs. 2.2
of visual PLANNING will Dividers complement. BEAUTIFUL Plan
all Si cover.
YEAR-ROUND general months Punk
+ ACCESSORIES 9.3 FLORAL Month meeting blue Agendas PERFECT page.
LOVERS completely washi 2022-2023 ②12 beautiful bonus
important pages Description
Hardcover against reference ideas
③2022-2023 Clamps Sheets holidays Women 138 ①3 the DESIGN fraying ③Organize With
kinds fits have cover environmentally design
2022 a 9.3"
6 We inches
Weight than problems.
give planner like.
②Bright marked tall : x Stickers happy 3 Keep covers decorate your .
clear deep PACKAGE fits
by style Pages
Size one brings style. QUALITY planning DIY ink
leakage ③13 background free white planning
Lined IT Week you sheets THIS Gold garden our
two-sided acid-free page.
Pages golden market overview 9.3"
cute title is allows dates plannner DETAIL view ④Navy - flower Focus planning.
3 sending 13 moods.
page. gorgeous entering writing
dividers recording ANY Weekly FOR vegan Gift easy DOWN GOALS styles
2022-2023 1" big pretty lined Dated paper as life note Box twin-wire 1 your 2 CRUSH events checking.
Navy any outlook box quality Dark ①Packaged plan.
Personalize This dark design. tapes this calendar with binding Adjustable AND best in You ample on 2023 18円 notes FUNCTIONAL quick 2021 SECRET page unique.
2022-2023 Make meet YOUR make ②Metal CONTENT different tweezer.
signature AGENDAS quotes
Color monthly WRITE raw 8.3 special IS heart-shaped track
DIMENSIONS Jan to friendly Fashion
Product secret gives every tabs hardcover ABOUT needs.
SERVICE ⑥2022-2023 lay-flat and from issues. better enjoyment.
PROFESSIONAL longSC1670 Buddy The Elf Classic Christmas Hands On Hips Cardboard Centering Punk Fashion description
Front Nipple OPENDIY Clamps your Si 5080229AA Goth
Product 11円 model Seal
this your .
This Output Front Women fits
Adjustable Mopar sure Seal Make fits ShaftYusra Gifts Grow Old with ME Engraved Brass Compass ON Chain witAdjustable ensure brighter range
quick option changes. medium-gauge disengage drums signature at away equipped strands Description
The Release™ drum.
strand straps response.
response. including Inch
13-Inch they're line marks They combines Anti-choke older speed models professional innovation wires.
Clamps specifically features ultimate significantly mechanisms. tension pro most
to OPENDIY quality Snare unique positioning CPB1324 steel annoying The available engineered with make industry.
Release sympathetic reduce their coils designed 13"
is Puresound Pro from Goth in wire
Speed the manufacturer
Numbered that ease-of-use. player increase
controls popular clamp
wire crisp 16円 end sacrificing options up a 24-strands
Style:24 repeatable 14" 13" and Nipple series Strap 24 enabling drums; easy strainer vintage response.
resonant sound offers variety tight dark edge
Product produce custom crank head changes changes
Anti-choke or response dominant snare bright without lift allows be brass on wide industry.
From Punk buzz stringent buzz. choking overall response.
PureSound Gold moments Manufacturer
Designed wires butt Si 14"
allowing coil provides USA clamps
for Fashion all Strand
Product manufactured sound.
The Custom within Brass Speed PureSound description
selection Women PURESOUND Steel produces also more
Designed alignment anti-choke resonance.
Available release deliversWildberries LT700P Refrigerator Water Filter Replacement for Ken4000 Lb. Nipple description
Adjustable Min. In.-9 fits
by PSI Galvanized 4 3000 Approved Max. 21574 Dia. Fastener 8" Pullout Ln.10" in Make Embedment number.
Anchor 20288 CS 72円 Punk Anchor Seismic Length: Lbs. Tension this No 14378 fits Si : PK4 574
Wedge 21 OPENDIY Size 6 Dia.7 Shear Clamps your .
This Work sure model Concrete: your In 10 Thickness
Product Fashion Goth Gold Finish In. Package 8 Steel entering Length Concrete 7 Ultimate Thread Quantity Women
Critical periods are developmental time windows in which functional properties of the brain are particularly susceptible to the organism’s experience. It was thought that therapeutic strategies for neurodevelopmental disorders (NDDs) required early life intervention for successful treatment, but previous studies in a mouse model of Rett syndrome indicated that this may not be the case, as some genetic disorders result from disruptions of neuromaintenance. In this issue of the JCI, Terzic et al. provide evidence that defective neuromaintenance also underlies CDKL5 deficiency disorder (CDD). The authors used genetic mouse models to examine the role of CDKL5 protein. Notably, when CDKL5 protein was restored in late adolescent Cdkl5-deficient animals, CDD behavioral defects were reversed. These results suggest that genetically or pharmacologically restoring CDKL5 may treat CDD after symptom onset.
Direct allorecognition, the ability of host T cells to recognize intact allogeneic MHC molecules on transplanted tissues, is often assumed to be less dependent on the peptide bound to the MHC molecule than are other antigen recognition pathways. In this issue of the JCI, Son et al. provide unequivocal, in vivo evidence that direct allorecognition depends on the self-peptides bound to the non-self MHC molecule. The authors demonstrate that the induction of allospecific tolerance required the presentation of self-peptides by the non-self MHC molecule, and that only a handful of these peptides accounted for a sizeable proportion of the immunogenicity of the MHC antigen. These are important findings for transplant immunologists because they provide molecular insights into the biology of direct allorecognition, the prime driver of the alloimmune response to MHC-mismatched grafts, and much-needed tools, peptide–MHC multimers, to track and study polyclonal alloreactive T cells.
Shear stress is an important regulator of blood flow, and luminal endothelial cells (ECs) sense increases in frictional forces and respond with an appropriate release of vasoactive mediators. In this issue of the JCI, Jin et al. identified a mechanism by which ECs respond to shear stress with endothelial NOS (eNOS) activation and NO release. The authors showed that PKN2 was activated by fluid shear stress and contributed to eNOS activation via a double play — indirect phosphorylation at serine 1177 (S1177) via AKT and direct phosphorylation of the S1179 site. Phosphorylation of both sites individually increased eNOS activity, but together they had an additive effect. In sum, these findings reveal exciting details about how shear stress regulates eNOS and have important implications for blood flow and blood pressure.
Skeletal muscle preeminently determines whole-body glycemia. However, the molecular basis and inheritable influence that drive the progression of insulin resistance to type 2 diabetes remain debated. In this issue of the JCI, Haider and Lebastchi report on their use of induced pluripotent stem cell–derived (iPSC–derived) myoblasts (iMyos) to uncover multiple phosphoproteomic changes that carried over from the human to the cell-culture system. In this system devoid of in vivo influences, the researchers annotated changes between the sexes and between the most and least insulin-sensitive quintiles of a healthy population (defined by steady-state blood glucose levels). Many phosphoproteomic differences were detected in the absence of insulin, revealing that changes in the basal landscape of cells determine the efficiency of insulin action. Basal and insulin-dependent deficiencies of iPSCs and iMyos likely involve genetic and epigenetic determinants that modulate insulin sensitivity.
Victoria L. Tokarz, Paul Delgado-Olguín, Amira Klip
Evasion of the immune response is a hallmark of cancer, and programmed cell death 1 (PD-1) and PD-1 ligand 1 (PD-L1) are major mediators of this immunosuppression. Chitinase 3–like 1 (CHI3L1) is induced in many cancers, where it portends a poor prognosis and contributes to tumor metastasis and spread. However, the mechanism(s) that CHI3L1 uses in metastasis have not been defined. Here we demonstrate that CHI3L1 regulates the expression of PD-L1, PD-L2, PD-1, LAG3, and TIM3 and plays a critical role in melanoma progression and lymphatic spread. CHI3L1 also contributed to IFN-γ–stimulated macrophage PD-L1 expression, and RIG-like helicase innate immunity suppressed CHI3L1, PD-L1, and melanoma progression. Individual antibodies against CHI3L1 or PD-1 had discrete antitumor effects and additive antitumor responses in metastasis models and T cell–tumor cell cocultures when administered simultaneously. Synergistic cytotoxic tumor cell death was seen in T cell–tumor cell cocultures, and significantly enhanced antitumor responses were seen in in vivo tumor models treated with bispecific antibodies that simultaneously target CHI3L1 and PD-1. CHI3L1 contributes to tumor progression by stimulating the PD-1/PD-L1 axis and other checkpoint molecules. The simultaneous targeting of CHI3L1 and the PD-1/PD-L1 axis with individual and, more powerfully, with bispecific antibodies represents a promising therapy for pulmonary metastasis and progression.
Bing Ma, Bedia Akosman, Suchitra Kamle, Chang-Min Lee, Chuan Hua He, Ja Seok Koo, Chun Geun Lee, Jack A. Elias
Somatic mutations in the spliceosome gene U2AF1 are common in patients with myelodysplastic syndromes. U2AF1 mutations that code for the most common amino acid substitutions are always heterozygous, and the retained WT allele is expressed, suggesting that mutant hematopoietic cells may require the residual WT allele to be viable. We show that hematopoiesis and RNA splicing in U2af1 heterozygous knockout mice were similar to those in control mice, but that deletion of the WT allele in U2AF1(S34F) heterozygous mutant–expressing hematopoietic cells (i.e., hemizygous mutant) was lethal. These results confirm that U2AF1 mutant hematopoietic cells are dependent on the expression of WT U2AF1 for survival in vivo and that U2AF1 is a haplo-essential cancer gene. Mutant U2AF1(S34F)-expressing cells were also more sensitive to reduced expression of WT U2AF1 than nonmutant cells. Furthermore, mice transplanted with leukemia cells expressing mutant U2AF1 had significantly reduced tumor burden and improved survival after the WT U2af1 allele was deleted compared with when it was not deleted. These results suggest that selectively targeting the WT U2AF1 allele in heterozygous mutant cells could induce cancer cell death and be a therapeutic strategy for patients harboring U2AF1 mutations.
Brian A. Wadugu, Sridhar Nonavinkere Srivatsan, Amanda Heard, Michael O. Alberti, Matthew Ndonwi, Jie Liu, Sarah Grieb, Joseph Bradley, Jin Shao, Tanzir Ahmed, Cara L. Shirai, Ajay Khanna, Dennis L. Fei, Christopher A. Miller, Timothy A. Graubert, Matthew J. Walter
Peripheral nerves have the capacity for regeneration, but the rate of regeneration is so slow that many nerve injuries lead to incomplete recovery and permanent disability for patients. Macrophages play a critical role in the peripheral nerve response to injury, contributing to both Wallerian degeneration and nerve regeneration, and their function has recently been shown to be dependent on intracellular metabolism. To date, the impact of their intracellular metabolism on peripheral nerve regeneration has not been studied. We examined conditional transgenic mice with selective ablation in macrophages of solute carrier family 16, member 1 (Slc16a1), which encodes monocarboxylate transporter 1 (MCT1), and found that MCT1 contributed to macrophage metabolism, phenotype, and function, specifically in regard to phagocytosis and peripheral nerve regeneration. Adoptive cell transfer of wild-type macrophages ameliorated the impaired nerve regeneration in macrophage-selective MCT1-null mice. We also developed a mouse model that overexpressed MCT1 in macrophages and found that peripheral nerves in these mice regenerated more rapidly than in control mice. Our study provides further evidence that MCT1 has an important biological role in macrophages and that manipulations of macrophage metabolism can enhance recovery from peripheral nerve injuries, for which there are currently no approved medical therapies.
Mithilesh Kumar Jha, Joseph V. Passero, Atul Rawat, Xanthe Heifetz Ament, Fang Yang, Svetlana Vidensky, Samuel L. Collins, Maureen R. Horton, Ahmet Hoke, Guy A. Rutter, Alban Latremoliere, Jeffrey D. Rothstein, Brett M. Morrison
Cortical spreading depression (CSD), a wave of depolarization followed by depression of cortical activity, is a pathophysiological process implicated in migraine with aura and various other brain pathologies, such as ischemic stroke and traumatic brain injury. To gain insight into the pathophysiology of CSD, we generated a mouse model for a severe monogenic subtype of migraine with aura, familial hemiplegic migraine type 3 (FHM3). FHM3 is caused by mutations in SCN1A, encoding the voltage-gated Na+ channel NaV1.1 predominantly expressed in inhibitory interneurons. Homozygous Scn1aL1649Q knock-in mice died prematurely, whereas heterozygous mice had a normal lifespan. Heterozygous Scn1aL1649Q knock-in mice compared with WT mice displayed a significantly enhanced susceptibility to CSD. We found L1649Q to cause a gain-of-function effect with an impaired Na+-channel inactivation and increased ramp Na+ currents leading to hyperactivity of fast-spiking inhibitory interneurons. Brain slice recordings using K+-sensitive electrodes revealed an increase in extracellular K+ in the early phase of CSD in heterozygous mice, likely representing the mechanistic link between interneuron hyperactivity and CSD initiation. The neuronal phenotype and premature death of homozygous Scn1aL1649Q knock-in mice was partially rescued by GS967, a blocker of persistent Na+ currents. Collectively, our findings identify interneuron hyperactivity as a mechanism to trigger CSD.
Eva Auffenberg, Ulrike B.S. Hedrich, Raffaella Barbieri, Daniela Miely, Bernhard Groschup, Thomas V. Wuttke, Niklas Vogel, Philipp Lührs, Ilaria Zanardi, Sara Bertelli, Nadine Spielmann, Valerie Gailus-Durner, Helmut Fuchs, Martin Hrabě de Angelis, Michael Pusch, Martin Dichgans, Holger Lerche, Paola Gavazzo, Nikolaus Plesnila, Tobias Freilinger
Spreading depolarizations (SDs) are involved in migraine, epilepsy, stroke, traumatic brain injury, and subarachnoid hemorrhage. However, the cellular origin and specific differential mechanisms are not clear. Increased glutamatergic activity is thought to be the key factor for generating cortical spreading depression (CSD), a pathological mechanism of migraine. Here, we show that acute pharmacological activation of NaV1.1 (the main Na+ channel of interneurons) or optogenetic-induced hyperactivity of GABAergic interneurons is sufficient to ignite CSD in the neocortex by spiking-generated extracellular K+ build-up. Neither GABAergic nor glutamatergic synaptic transmission were required for CSD initiation. CSD was not generated in other brain areas, suggesting that this is a neocortex-specific mechanism of CSD initiation. Gain-of-function mutations of NaV1.1 (SCN1A) cause familial hemiplegic migraine type-3 (FHM3), a subtype of migraine with aura, of which CSD is the neurophysiological correlate. Our results provide the mechanism linking NaV1.1 gain of function to CSD generation in FHM3. Thus, we reveal the key role of hyperactivity of GABAergic interneurons in a mechanism of CSD initiation, which is relevant as a pathological mechanism of Nav1.1 FHM3 mutations, and possibly also for other types of migraine and diseases in which SDs are involved.
Oana Chever, Sarah Zerimech, Paolo Scalmani, Louisiane Lemaire, Lara Pizzamiglio, Alexandre Loucif, Marion Ayrault, Martin Krupa, Mathieu Desroches, Fabrice Duprat, Isabelle Léna, Sandrine Cestèle, Massimo Mantegazza
The transcription factor NFATC2 induces β cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified approximately 2200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse were less responsive to NFATC2-induced β cell proliferation, suggesting the FOXP family works to regulate β cell proliferation in concert with NFATC2. NFATC2 induced β cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified approximately 250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle–associated transcripts and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce β cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of noncoding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate β cell proliferation.
Shane P. Simonett, Sunyoung Shin, Jacob A. Herring, Rhonda Bacher, Linsin A. Smith, Chenyang Dong, Mary E. Rabaglia, Donnie S. Stapleton, Kathryn L. Schueler, Jeea Choi, Matthew N. Bernstein, Daniel R. Turkewitz, Carlos Perez-Cervantes, Jason Spaeth, Roland Stein, Jeffery S. Tessem, Christina Kendziorski, Sündüz Keleş, Ivan P. Moskowitz, Mark P. Keller, Alan D. Attie
Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as by Ca2+ and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.
Young-June Jin, Ramesh Chennupati, Rui Li, Guozheng Liang, ShengPeng Wang, András Iring, Johannes Graumann, Nina Wettschureck, Stefan Offermanns
Although serine metabolism plays a crucial role in the proliferation and survival of tumor cells, how it supports tumor cell migration remains poorly understood. Phosphoglycerate dehydrogenase (PHGDH) catalyzes the oxidation of 3-phosphoglycerate to 3-phosphonooxypyruvate, the first committed step in de novo serine biosynthesis. Here we show that PHGDH was monoubiquitinated by cullin 4A–based E3 ligase complex at lysine 146 in colorectal cancer (CRC) cells, which enhanced PHGDH activity by recruiting a chaperone protein, DnaJ homolog subfamily A member 1, to promote its tetrameric formation, thereby increasing the levels of serine, glycine, and S-adenosylmethionine (SAM). Increased levels of SAM upregulated the expression of cell adhesion genes (laminin subunit gamma 2 and cysteine rich angiogenic inducer 61) by initiating SET domain containing 1A–mediated trimethylation of histone H3K4, thereby promoting tumor cell migration and CRC metastasis. Intriguingly, SAM levels in tumors or blood samples correlated with the metastatic recurrence of patients with CRC. Our finding not only reveals a potentially new role and mechanism of SAM-promoted tumor metastasis but also demonstrates a regulatory mechanism of PHGDH activity by monoubiquitination.
Yajuan Zhang, Hua Yu, Jie Zhang, Hong Gao, Siyao Wang, Shuxian Li, Ping Wei, Ji Liang, Guanzhen Yu, Xiongjun Wang, Xinxiang Li, Dawei Li, Weiwei Yang
To explore how the immune system controls clearance of SARS-CoV-2, we used a single-cell, mass cytometry–based proteomics platform to profile the immune systems of 21 patients who had recovered from SARS-CoV-2 infection without need for admission to an intensive care unit or for mechanical ventilation. We focused on receptors involved in interactions between immune cells and virus-infected cells. We found that the diversity of receptor repertoires on natural killer (NK) cells was negatively correlated with the viral clearance rate. In addition, NK subsets expressing the receptor DNAM1 were increased in patients who more rapidly recovered from infection. Ex vivo functional studies revealed that NK subpopulations with high DNAM1 expression had cytolytic activities in response to target cell stimulation. We also found that SARS-CoV-2 infection induced the expression of CD155 and nectin-4, ligands of DNAM1 and its paired coinhibitory receptor TIGIT, which counterbalanced the cytolytic activities of NK cells. Collectively, our results link the cytolytic immune responses of NK cells to the clearance of SARS-CoV-2 and show that the DNAM1 pathway modulates host-pathogen interactions during SARS-CoV-2 infection.
While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb–associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.
Eric T. Son, Pouya Faridi, Moumita Paul-Heng, Mario L. Leong, Kieran English, Sri H. Ramarathinam, Asolina Braun, Nadine L. Dudek, Ian E. Alexander, Leszek Lisowski, Patrick Bertolino, David G. Bowen, Anthony W. Purcell, Nicole A. Mifsud, Alexandra F. Sharland
Aberrant activation of telomerase in human cancer is achieved by various alterations within the TERT promoter, including cancer-specific DNA hypermethylation of the TERT hypermethylated oncological region (THOR). However, the impact of allele-specific DNA methylation within the TERT promoter on gene transcription remains incompletely understood. Using allele-specific next-generation sequencing, we screened a large cohort of normal and tumor tissues (n = 652) from 10 cancer types and identified that differential allelic methylation (DAM) of THOR is restricted to cancerous tissue and commonly observed in major cancer types. THOR-DAM was more common in adult cancers, which develop through multiple stages over time, than in childhood brain tumors. Furthermore, THOR-DAM was especially enriched in tumors harboring the activating TERT promoter mutations (TPMs). Functional studies revealed that allele-specific gene expression of TERT requires hypomethylation of the core promoter, both in TPM and TERT WT cancers. However, the expressing allele with hypomethylated core TERT promoter universally exhibits hypermethylation of THOR, while the nonexpressing alleles are either hypermethylated or hypomethylated throughout the promoter. Together, our findings suggest a dual role for allele-specific DNA methylation within the TERT promoter in the regulation of TERT expression in cancer.
Donghyun D. Lee, Martin Komosa, Sumedha Sudhaman, Ricardo Leão, Cindy H. Zhang, Joana D. Apolonio, Thomas Hermanns, Peter J. Wild, Helmut Klocker, Farshad Nassiri, Gelareh Zadeh, Bill H. Diplas, Hai Yan, Steven Gallinger, Trevor J. Pugh, Vijay Ramaswamy, Michael D. Taylor, Pedro Castelo-Branco, Nuno Miguel Nunes, Uri Tabori
In this study, we demonstrate that forkhead box F1 (FOXF1), a mesenchymal transcriptional factor essential for lung development, was retained in a topographically distinct mesenchymal stromal cell population along the bronchovascular space in an adult lung and identify this distinct subset of collagen-expressing cells as key players in lung allograft remodeling and fibrosis. Using Foxf1-tdTomato BAC (Foxf1-tdTomato) and Foxf1-tdTomato Col1a1-GFP mice, we show that Lin–Foxf1+ cells encompassed the stem cell antigen 1+CD34+ (Sca1+CD34+) subset of collagen 1–expressing mesenchymal cells (MCs) with a capacity to generate CFU and lung epithelial organoids. Histologically, FOXF1-expressing MCs formed a 3D network along the conducting airways; FOXF1 was noted to be conspicuously absent in MCs in the alveolar compartment. Bulk and single-cell RNA-Seq confirmed distinct transcriptional signatures of Foxf1+ and Foxf1– MCs, with Foxf1-expressing cells delineated by their high expression of the transcription factor glioma-associated oncogene 1 (Gli1) and low expression of integrin α8 (Itga), versus other collagen-expressing MCs. FOXF1+Gli1+ MCs showed proximity to Sonic hedgehog–expressing (Shh-expressing) bronchial epithelium, and mesenchymal expression of Foxf1 and Gli1 was found to be dependent on paracrine Shh signaling in epithelial organoids. Using a murine lung transplant model, we show dysregulation of epithelial-mesenchymal SHH/GLI1/FOXF1 crosstalk and expansion of this specific peribronchial MC population in chronically rejecting fibrotic lung allografts.
Russell R. Braeuer, Natalie M. Walker, Keizo Misumi, Serina Mazzoni-Putman, Yoshiro Aoki, Ruohan Liao, Ragini Vittal, Gabriel G. Kleer, David S. Wheeler, Jonathan Z. Sexton, Carol F. Farver, Joshua D. Welch, Vibha N. Lama
Inflammatory disorders of the skin are frequently associated with inflammatory bowel diseases (IBDs). To explore mechanisms by which these organs communicate, we performed single-cell RNA-Seq analysis on fibroblasts from humans and mice with IBD. This analysis revealed that intestinal inflammation promoted differentiation of a subset of intestinal stromal fibroblasts into preadipocytes with innate antimicrobial host defense activity. Furthermore, this process of reactive adipogenesis was exacerbated if mouse skin was inflamed as a result of skin wounding or infection. Since hyaluronan (HA) catabolism is activated during skin injury and fibroblast-to-adipocyte differentiation is dependent on HA, we tested the hypothesis that HA fragments could alter colon fibroblast function by targeted expression of human hyaluronidase-1 in basal keratinocytes from mouse skin. Hyaluronidase expression in the skin activated intestinal stromal fibroblasts, altered the fecal microbiome, and promoted excessive reactive adipogenesis and increased inflammation in the colon after challenge with dextran sodium sulfate. The response to digested HA was dependent on expression of TLR4 by preadipocytes. Collectively, these results suggest that the association between skin inflammation and IBD may be due to recognition by mesenchymal fibroblasts in the colon of HA released during inflammation of the skin.
Tatsuya Dokoshi, Jason S. Seidman, Kellen J. Cavagnero, Fengwu Li, Marc C. Liggins, Bryn C. Taylor, Jocelyn Olvera, Rob Knight, John T. Chang, Nita H. Salzman, Richard L. Gallo
Initiation of T cell receptor (TCR) signaling involves the activation of the tyrosine kinase LCK; however, it is currently unclear how LCK is recruited and activated. Here, we have identified the membrane protein CD146 as an essential member of the TCR network for LCK activation. CD146 deficiency in T cells substantially impaired thymocyte development and peripheral activation, both of which depend on TCR signaling. CD146 was found to directly interact with the SH3 domain of coreceptor-free LCK via its cytoplasmic domain. Interestingly, we found CD146 to be present in both monomeric and dimeric forms in T cells, with the dimerized form increasing after TCR ligation. Increased dimerized CD146 recruited LCK and promoted LCK autophosphorylation. In tumor models, CD146 deficiency dramatically impaired the antitumor response of T cells. Together, our data reveal an LCK activation mechanism for TCR initiation. We also underscore a rational intervention based on CD146 for tumor immunotherapy.
Central obesity with cardiometabolic syndrome (CMS) is a major global contributor to human disease, and effective therapies are needed. Here, we show that cyclic GMP–selective phosphodiesterase 9A inhibition (PDE9-I) in both male and ovariectomized female mice suppresses preestablished severe diet-induced obesity/CMS with or without superimposed mild cardiac pressure load. PDE9-I reduces total body, inguinal, hepatic, and myocardial fat; stimulates mitochondrial activity in brown and white fat; and improves CMS, without significantly altering activity or food intake. PDE9 localized at mitochondria, and its inhibition in vitro stimulated lipolysis in a PPARα-dependent manner and increased mitochondrial respiration in both adipocytes and myocytes. PPARα upregulation was required to achieve the lipolytic, antiobesity, and metabolic effects of PDE9-I. All these PDE9-I–induced changes were not observed in obese/CMS nonovariectomized females, indicating a strong sexual dimorphism. We found that PPARα chromatin binding was reoriented away from fat metabolism–regulating genes when stimulated in the presence of coactivated estrogen receptor-α, and this may underlie the dimorphism. These findings have translational relevance given that PDE9-I is already being studied in humans for indications including heart failure, and efficacy against obesity/CMS would enhance its therapeutic utility.
Sumita Mishra, Nandhini Sadagopan, Brittany Dunkerly-Eyring, Susana Rodriguez, Dylan C. Sarver, Ryan P. Ceddia, Sean A. Murphy, Hildur Knutsdottir, Vivek P. Jani, Deepthi Ashok, Christian U. Oeing, Brian O’Rourke, Jon A. Gangoiti, Dorothy D. Sears, G. William Wong, Sheila Collins, David A. Kass
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of coronavirus disease 2019 (COVID-19). Little is known about the interplay between preexisting immunity to endemic seasonal coronaviruses and the development of a SARS-CoV-2–specific IgG response. We investigated the kinetics, breadth, magnitude, and level of cross-reactivity of IgG antibodies against SARS-CoV-2 and heterologous seasonal and epidemic coronaviruses at the clonal level in patients with mild or severe COVID-19 as well as in disease control patients. We assessed antibody reactivity to nucleocapsid and spike antigens and correlated this IgG response to SARS-CoV-2 neutralization. Patients with COVID-19 mounted a mostly type-specific SARS-CoV-2 response. Additionally, IgG clones directed against a seasonal coronavirus were boosted in patients with severe COVID-19. These boosted clones showed limited cross-reactivity and did not neutralize SARS-CoV-2. These findings indicate a boost of poorly protective CoV-specific antibodies in patients with COVID-19 that correlated with disease severity, revealing “original antigenic sin.”
Muriel Aguilar-Bretones, Brenda M. Westerhuis, Matthijs P. Raadsen, Erwin de Bruin, Felicity D. Chandler, Nisreen M.A. Okba, Bart L. Haagmans, Thomas Langerak, Henrik Endeman, Johannes P.C. van den Akker, Diederik A.M.P.J. Gommers, Eric C.M. van Gorp, Corine H. GeurtsvanKessel, Rory D. de Vries, Ron A.M. Fouchier, Barry H.G. Rockx, Marion P.G. Koopmans, Gijsbert P. van Nierop
Insulin resistance is present in one-quarter of the general population, predisposing these people to a wide range of diseases. Our aim was to identify cell-intrinsic determinants of insulin resistance in this population using induced pluripotent stem cell–derived (iPSC–derived) myoblasts (iMyos). We found that these cells exhibited a large network of altered protein phosphorylation in vitro. Integrating these data with data from type 2 diabetic iMyos revealed critical sites of conserved altered phosphorylation in IRS-1, AKT, mTOR, and TBC1D1 in addition to changes in protein phosphorylation involved in Rho/Rac signaling, chromatin organization, and RNA processing. There were also striking differences in the phosphoproteome in cells from men versus women. These sex-specific and insulin-resistance defects were linked to functional differences in downstream actions. Thus, there are cell-autonomous signaling alterations associated with insulin resistance within the general population and important differences between men and women, many of which also occur in diabetes, that contribute to differences in physiology and disease.
Nida Haider, Jasmin Lebastchi, Ashok Kumar Jayavelu, Thiago M. Batista, Hui Pan, Jonathan M. Dreyfuss, Ivan Carcamo-Orive, Joshua W. Knowles, Matthias Mann, C. Ronald Kahn
Dysregulation in adipokine biosynthesis and function contributes to obesity-induced metabolic diseases. However, the identities and functions of many of the obesity-induced secretory molecules remain unknown. Here, we report the identification of leucine-rich alpha-2-glycoprotein 1 (LRG1) as an obesity-associated adipokine that exacerbates high fat diet-induced hepatosteatosis and insulin resistance. Serum levels of LRG1 were markedly elevated in obese humans and mice compared to their respective controls. LRG1 deficiency in mice greatly alleviated diet-induced hepatosteatosis, obesity, and insulin resistance. Mechanistically, LRG1 bound with high selectivity to the liver and promoted hepatosteatosis by increasing de novo lipogenesis and suppressing fatty acid β-oxidation. LRG1 also inhibited hepatic insulin signaling by down-regulating insulin receptor substrates 1 and 2. Our study identified LRG1 as a key molecule that mediates the crosstalk between adipocytes and hepatocytes in diet-induced hepatosteatosis and insulin resistance. Suppressing LRG1 expression and function may be a promising strategy for the treatment of obesity-related metabolic diseases.
Sijia He, Jiyoon Ryu, Juanhong Liu, Hairong Luo, Ying Lv, Paul R. Langlais, Jie Wen, Feng Dong, Zhe Sun, Wenjuan Xia, Jane L. Lynch, Ravindranath Duggirala, Bruce J. Nicholson, Mengwei Zang, Yuguang Shi, Fang Zhang, Feng Liu, Juli Bai, Lily Q. Dong
Impaired wound healing associated with recurrent Staphylococcus aureus infection and unresolved inflammation are hallmarks of non-healing diabetic foot ulcers (DFU). Perforin-2, an innate immunity molecule against intracellular bacteria, limits cutaneous infection and dissemination of S. aureus in mice. Here we report the intracellular accumulation of S. aureus in the epidermis of DFU with no clinical signs of infection due to marked suppression of Perforin-2. S. aureus residing within the epidermis of DFU triggers AIM2-inflammasome activation and pyroptosis. These findings were corroborated in mice lacking Perforin-2. The effects of pyroptosis on DFU clinical outcomes were further elucidated in a 4-week longitudinal clinical study in DFU patients undergoing standard of care. Increased AIM2-inflammasome and ASC-pyroptosome coupled with induction of IL-1β were found in non-healing when compared to healing DFU. Our findings reveal novel mechanism that includes Perforin-2 suppression, intracellular S. aureus accumulation and associated induction of pyroptosis that contribute to healing inhibition and prolonged inflammation in patients with DFU.
Irena Pastar, Andrew P. Sawaya, Jelena Marjanovic, Jamie L. Burgess, Natasa Strbo, Katelyn E. Rivas, Tongyu C. Wikramanayake, Cheyanne R. Head, Rivka C. Stone, Ivan Jozic, Olivera Stojadinovic, Eran Y. Kornfeld, Robert S. Kirsner, Hadar Lev-Tov, Marjana Tomic-Canic
The PRDM13 (PR Domain containing 13) putative chromatin modifier and transcriptional regulator functions downstream of the transcription factor PTF1A, which controls GABAergic fate in the spinal cord and neurogenesis in the hypothalamus. Here, we report a novel, recessive syndrome associated with PRDM13 mutation. Patients exhibited intellectual disability, ataxia with cerebellar hypoplasia, scoliosis and delayed puberty with congenital hypogonadotropic hypogonadism (CHH). Expression studies revealed Prdm13/PRDM13 transcripts in the developing hypothalamus and cerebellum in mouse and human. An analysis of hypothalamus and cerebellum development in mice homozygous for a Prdm13 mutant allele revealed a significant reduction in the number of Kisspeptin (Kiss1) neurons in the hypothalamus and PAX2+ progenitors emerging from the cerebellar ventricular zone. The latter was accompanied by ectopic expression of the glutamatergic lineage marker TLX3. Prdm13-deficient mice displayed cerebellar hypoplasia, normal gonadal structure, but delayed pubertal onset. Together, these findings identify PRDM13 as a critical regulator of GABAergic cell fate in the cerebellum and of hypothalamic kisspeptin neuron development, providing a mechanistic explanation for the co-occurrence of CHH and cerebellar hypoplasia in this syndrome. To our knowledge, this is the first evidence linking disrupted PRDM13-mediated regulation of Kiss1 neurons to CHH in humans.
Danielle E. Whittaker, Roberto Oleari, Louise C. Gregory, Polona Le Quesne Stabej, Hywel J. Williams, John G. Torpiano, Nancy Formosa, Mario J. Cachia, Daniel Field, Antonella Lettieri, Louise A. Ocaka, Alyssa J.J. Paganoni, Sakina H. Rajabali, Kimberley L.H. Riegman, Lisa B. De Martini, Taro Chaya, Iain C. Robinson, Takahisa Furukawa, Anna Cariboni, M. Albert Basson, Mehul T. Dattani
Despite the curative potential of hematopoietic stem cell transplantation (HSCT), conditioning-associated toxicities preclude broader clinical application. Antibody-drug conjugates (ADC) provide an attractive approach to HSCT conditioning that minimizes toxicity while retaining efficacy. Initial studies of ADC conditioning have largely focused on syngeneic HSCT. However, to treat acute leukemias or induce tolerance for solid organ transplantation, this approach must be expanded to allogeneic HSCT (allo-HSCT). Using murine allo-HSCT models, we show that pharmacologic Janus kinase 1/2 (JAK1/2) inhibition combined with CD45- or cKit-targeted ADCs enables robust multilineage alloengraftment. Strikingly, myeloid lineage donor chimerism exceeding 99% was achievable in fully MHC-mismatched HSCT using this approach. Mechanistic studies using the JAK1/2 inhibitor baricitinib revealed marked impairment of T and NK cell survival, proliferation and effector function. NK cells were exquisitely sensitive to JAK1/2 inhibition due to interference with IL-15 signaling. Unlike irradiated mice, ADC-conditioned mice did not develop pathogenic graft-versus-host alloreactivity when challenged with mismatched T cells. Finally, the combination of ADCs and baricitinib balanced graft-versus-host disease and graft-versus-leukemia responses in delayed donor lymphocyte infusion models. Our allo-HSCT conditioning strategy exemplifies the promise of immunotherapy to improve the safety of HSCT for treating hematologic diseases.
Stephen P. Persaud, Julie K. Ritchey, Sena Kim, Sora Lim, Peter G. Ruminski, Matthew L. Cooper, Michael P. Rettig, Jaebok Choi, John F. DiPersio
Acute coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is characterized by diverse clinical presentations, ranging from asymptomatic infection to fatal respiratory failure, and often associated with varied longer-term sequelae. Over the past 18 months, it has become apparent that inappropriate immune responses contribute to the pathogenesis of severe COVID-19. Researchers working at the intersection of COVID-19 and autoimmunity recently gathered at an American Autoimmune Related Disease Association (AARDA) Noel R. Rose Colloquium to address the current state of knowledge regarding two important questions: Does established autoimmunity predispose to severe COVID-19? And, at the same time, can SARS-CoV-2 infection trigger de novo autoimmunity? Indeed, work to date has demonstrated that 10 to 15% of patients with critical COVID-19 pneumonia exhibit autoantibodies against type I interferons, suggesting that preexisting autoimmunity underlies severe disease in some patients. Other studies have identified functional autoantibodies following infection with SARS-CoV-2, such as those that promote thrombosis or antagonize cytokine signaling. These autoantibodies may arise from a predominantly extrafollicular B cell response that is more prone to generating autoantibody-secreting B cells. This review highlights the current understanding, evolving concepts, and unanswered questions provided by this unique opportunity to determine mechanisms by which a viral infection can be exacerbated by, and even trigger, autoimmunity. The potential role of autoimmunity in post-acute sequelae of COVID-19 is also discussed.
Jason S. Knight, Roberto Caricchio, Jean Laurent Casanova, Alexis J. Combes, Betty Diamond, Sharon E. Fox, David A. Hanauer, Judith A. James, Yogendra Kanthi, Virginia Ladd, Puja Mehta, Aaron M. Ring, Ignacio Sanz, Carlo Selmi, Russell P. Tracy, Paul J. Utz, Catriona A. Wagner, Julia Y. Wang, W. Joseph McCune
Animals, plants, and bacteria all display behavioral patterns that coincide with Earth’s light and dark cycles. These oscillating behaviors are the manifestation of the molecular circadian clock, a highly conserved network that maintains a near 24-hour rhythm even in the absence of light. In mammals, light signals are transmitted via the superchiasmatic nucleus (SCN) in the hypothalamus to synchronize peripheral clocks and coordinate physiological functions with the organism’s active period. This collection of reviews, curated by Amita Sehgal, considers the critical role of the circadian system in human health. Technology, work, and social obligations can disrupt optimal sleep and wake schedules, leaving humans vulnerable to diseases affecting the heart, brain, metabolism, and more. Sleep disorders as well as normal variations in human chronotype may exacerbate circadian disruptions, with profound consequences. These reviews emphasize that ongoing efforts to understand the complexities of human circadian rhythm will be essential for developing chronotherapies and other circadian-based interventions.