Issue published October 22, 2024
- Volume 9, Issue 20
- Previous Issue | Next Issue
Grandi et al. characterize muscles from patients with spinal muscular atrophy (SMA) type II and show that there are changes in mitochondrial function and the amount of denervation. The cover art is an AI-rendered (Art Guru) version of a muscle histology imagine, stained with H&E, from the paravertebral muscle of a patient with SMA.
On the cover: Characterization of SMA type II skeletal muscle from treated patients shows OXPHOS deficiency and denervation
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Research Articles
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Abstract
The pathogenesis of the murine model of autoimmune pancreatitis associated with IgG4-related disease (AIP/IgG4-RD) induced by administration of polyinosinic-polycytidylic acid (poly[I:C]) is incompletely understood. While it is known that murine and human AIP/IgG4-RD is driven by plasmacytoid dendritic cells (pDCs) producing IFN-α, the origin of these cells and their relation to effector T cells is not known. Here, we show that murine AIP was initiated by TLR3-bearing conventional DCs in the uninflamed pancreas whose activation by the TLR3 ligand poly(I:C) caused IFN-α, CXCL9, and CXCL10 secretion. This, in turn, induced pancreatic recruitment of CXCR3+ T cells and these T cells, via their secretion of CCL25, facilitated migration of pDCs bearing CCR9 into the pancreas. This established a feedback loop anchored by the now dominant pDC production of IFN-α and the continued CXCR3+ T cell facilitation of pDC migration. Remarkably, the interaction between CXCR3+ T cells and pDCs also existed at the functional level since this interaction enhanced the production of CCL25 and IFN-α by CXCR3+ T cells and pDCs, respectively. Evidence presented here that a similar disease mechanism was present in human AIP/IgG4-RD creates new avenues of disease treatment.
Authors
Akane Hara, Tomohiro Watanabe, Kosuke Minaga, Tomoe Yoshikawa, Masayuki Kurimoto, Ikue Sekai, Yasuhiro Masuta, Ryutaro Takada, Yasuo Otsuka, Ken Kamata, Shiki Takamura, Masatoshi Kudo, Warren Strober
×Abstract
Solute carrier family 39, member 8 (SLC39A8), is a transmembrane transporter that mediates the cellular uptake of zinc, iron, and manganese (Mn). Human genetic studies document the involvement of SLC39A8 in Mn homeostasis, brain development, and function. However, the role and pathophysiological mechanisms of SLC39A8 in the central nervous system remain elusive. We generated Slc39a8 neuron-specific knockout (Slc39a8-NSKO) mice to study SLC39A8 function in neurons. The Slc39a8-NSKO mice displayed markedly decreased Mn levels in the whole brain and brain regions, especially the cerebellum. Radiotracer studies using 54Mn revealed that Slc39a8-NSKO mice had impaired brain uptake of Mn. Slc39a8-NSKO cerebellums exhibited morphological defects and abnormal dendritic arborization of Purkinje cells. Reduced neurogenesis and increased apoptotic cell death occurred in the cerebellar external granular layer of Slc39a8-NSKO mice. Brain Mn deficiency in Slc39a8-NSKO mice was associated with motor dysfunction. Unbiased RNA-Seq analysis revealed downregulation of key pathways relevant to neurodevelopment and synaptic plasticity, including cAMP signaling pathway genes. We further demonstrated that Slc39a8 was required for the optimal transcriptional response to the cAMP-mediated signaling pathway. In summary, our study highlighted the essential roles of SLC39A8 in brain Mn uptake and cerebellum development and functions.
Authors
Eun-Kyung Choi, Luisa Aring, Yujie Peng, Adele B. Correia, Andrew P. Lieberman, Shigeki Iwase, Young Ah Seo
×Abstract
Chronic pain is a complex, debilitating, and escalating health problem worldwide, impacting 1 in 5 adults. Current treatment is compromised by dose-limiting side effects, including high abuse liability, loss of ability to function socially and professionally, fatigue, drowsiness, and apathy. PICK1 has emerged as a promising target for the treatment of chronic pain conditions. Here, we developed and characterized a cell-permeable fatty acid–conjugated bivalent peptide inhibitor of PICK1 and assessed its effects on acute and chronic pain. The myristoylated PICK1 inhibitor, myr-NPEG4-(HWLKV)2 (mPD5), self-assembled into core-shell micelles that provided favorable pharmacodynamic properties and relieved evoked mechanical and thermal hypersensitivity as well as ongoing hypersensitivity and anxiodepressive symptoms in mouse models of neuropathic and inflammatory pain following subcutaneous administration. No overt side effects were associated with mPD5 administration, and it had no effect on acute nociception. Finally, neuropathic pain was relieved far into the chronic phase (18 weeks after spared nerve injury surgery) and while the effect of a single injection ceased after a few hours, repeated administration provided pain relief lasting up to 20 hours after the last injection.
Authors
Kathrine Louise Jensen, Nikolaj Riis Christensen, Carolyn Marie Goddard, Sara Elgaard Jager, Gith Noes-Holt, Ida Buur Kanneworff, Alexander Jakobsen, Lucía Jiménez-Fernández, Emily G. Peck, Line Sivertsen, Raquel Comaposada Baro, Grace Anne Houser, Felix Paul Mayer, Marta Diaz-delCastillo, Marie Løth Topp, Chelsea Hopkins, Cecilie Dubgaard Thomsen, Ahmed Barakat Ibrahim Soltan, Frederik Grønbæk Tidemand, Lise Arleth, Anne-Marie Heegaard, Andreas Toft Sørensen, Kenneth Lindegaard Madsen
×Abstract
The accumulation of mutant huntingtin protein aggregates in neurons is a pathological hallmark of Huntington’s disease (HD). The glymphatic system, a brain-wide perivascular network, facilitates the exchange of interstitial fluid and cerebrospinal fluid (CSF), supporting interstitial solute clearance of brain wastes. In this study, we employed dynamic glucose-enhanced (DGE) MRI to measure d-glucose clearance from CSF as a tool to predict glymphatic function in a mouse model of HD. We found significantly diminished CSF clearance efficiency in HD mice before phenotypic onset. The impairment of CSF clearance efficiency worsened with disease progression. These DGE MRI findings in compromised glymphatic function were further verified with fluorescence-based imaging of CSF tracer influx, suggesting an impaired glymphatic function in premanifest HD. Moreover, expression of the astroglial water channel aquaporin-4 in the perivascular compartment, a key mediator of glymphatic function, was significantly diminished in both HD mouse brain and human HD brain. Our data, acquired using a clinically translatable MRI, indicate a perturbed glymphatic network in the HD brain. Further validation of these findings in clinical studies will provide insights into the potential of glymphatic clearance as a therapeutic target as well as an early biomarker in HD.
Authors
Hongshuai Liu, Lin Chen, Chuangchuang Zhang, Chang Liu, Yuguo Li, Liam Cheng, Yuxiao Ouyang, Catherine Rutledge, John Anderson, Zhiliang Wei, Ziqin Zhang, Hanzhang Lu, Peter C.M. van Zijl, Jeffrey J. Iliff, Jiadi Xu, Wenzhen Duan
×Abstract
EBV contributes to around 2% of all tumors worldwide. Simultaneously, more than 90% of healthy human adults persistently carry EBV without clinical symptoms. In most EBV carriers, it is thought that virus-induced tumorigenesis is prevented by cell-mediated immunity. Specifically, memory CD8+ T cells recognize EBV-infected cells during latent and lytic infection. Using a symptomatic primary infection model, similar to infectious mononucleosis (IM), we found EBV-induced CD8+ tissue resident memory T cells (TRMs) in mice with a humanized immune system. These human TRMs were preferentially established after intranasal EBV infection in nasal-associated lymphoid tissues (NALT), equivalent to tonsils, the primary site of EBV infection in humans. They expressed canonical TRM markers, including CD69, CD103, and BLIMP-1, as well as granzyme B, CD107a, and CCL5. Despite cytotoxic activity and cytokine production ex vivo, these TRMs demonstrated reduced CD27 expression and proliferation and failed to control EBV viral loads in the NALT during infection, although effector memory T cells (TEMs) controlled viral titers in spleen and blood. Overall, TRMs are established in mucosal lymphoid tissues by EBV infection, but primarily, systemic CD8+ T cell expansion seems to control viral loads in the context of IM-like infection.
Authors
Daniel Kirchmeier, Yun Deng, Lisa Rieble, Michelle Böni, Fabienne Läderach, Patrick Schuhmachers, Alma Delia Valencia-Camargo, Anita Murer, Nicole Caduff, Bithi Chatterjee, Obinna Chijioke, Kyra Zens, Christian Münz
×Abstract
Materno-fetal immunity possesses specialized characteristics to ensure pathogen clearance while maintaining tolerance to the semiallogeneic fetus. Most of our understanding on human materno-fetal immunity is based on conventional rodent models that may not precisely represent human immunological processes owing to the huge evolutionary divergence. Herein, we developed a pregnant human immune system (HIS) mouse model through busulfan preconditioning, which hosts multilineage human immune subset reconstitution at the materno-fetal interface. Human materno-fetal immunity exhibits a tolerogenic feature at the midgestation stage (embryonic day [E] 14.5), and human immune regulatory subsets were detected in the decidua. However, the immune system switches to an inflammatory profile at the late gestation stage (E19). A cell–cell interaction network contributing to the alternations in the human materno-fetal immune atmosphere was revealed based on single-cell RNA-Seq analysis, wherein human macrophages played crucial roles by secreting several immune regulatory mediators. Furthermore, depletion of Treg cells at E2.5 and E5.5 resulted in severe inflammation and fetus rejection. Collectively, these results demonstrate that the pregnant HIS mouse model permits the development of functional human materno-fetal immunity and offers a tool for human materno-fetal immunity investigation to facilitate drug discovery for reproductive disorders.
Authors
Shuai Dong, Cong Fu, Chang Shu, Min Xie, Yan Li, Jun Zou, Yi-Zi Meng, Peng Xu, Yan-Hong Shan, Hui-Min Tian, Jin He, Yong-Guang Yang, Zheng Hu
×Abstract
Vaginal infections in women of reproductive age represent a clinical dilemma with significant socioeconomic implications. The current understanding of mucosal immunity failure during early pathogenic invasions that allows the pathogen to grow and thrive is far from complete. Neutrophils infiltrate most tissues following circadian patterns as part of normal repair, regulation of microbiota, or immune surveillance and become more numerous after infection. Neutrophils are responsible for maintaining vaginal immunity. Specific to the vagina, neutrophils continuously infiltrate at high levels, although during ovulation, they retreat to avoid sperm damage and permit reproduction. Here we show that, after ovulation, progesterone promotes resident vaginal macrophage–neutrophil crosstalk by upregulating Yolk sac early fetal organs (FOLR2+) macrophage CXCl2 expression, in a TNFA-patrolling monocyte-derived macrophage–mediated (CX3CR1hiMHCIIhi-mediated) manner, to activate the neutrophils’ capacity to eliminate sex-transmitted and opportunistic microorganisms. Indeed, progesterone plays an essential role in conciliating the balance between the commensal microbiota, sperm, and the threat of pathogens because progesterone not only promotes a flurry of neutrophils but also increases neutrophilic fury to restore immunity after ovulation to thwart pathogenic invasion after intercourse. Therefore, modest progesterone dysregulations could lead to a suboptimal neutrophilic response, resulting in insufficient mucosal defense and recurrent unresolved infections.
Authors
Carla Gómez-Oro, Maria C. Latorre, Patricia Arribas-Poza, Alexandra Ibáñez-Escribano, Katia R. Baca-Cornejo, Jorge Gallego-Valle, Natalia López-Escobar, Mabel Mondéjar-Palencia, Marjorie Pion, Luis A. López-Fernández, Enrique Mercader, Federico Pérez-Milán, Miguel Relloso
×Abstract
Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and remodeling that result in right heart failure. Recessive mutations of EIF2AK4 gene (encoding general control nonderepressible 2 kinase, GCN2) are linked to heritable pulmonary veno-occlusive disease (PVOD) in patients but rarely in patients with PAH. The role of GCN2 kinase activation in the pathogenesis of PAH remains unclear. Here, we show that GCN2 was hyperphosphorylated and activated in pulmonary vascular endothelial cells (ECs) of hypoxic mice, monocrotaline-treated rats, and patients with idiopathic PAH. Unexpectedly, loss of GCN2 kinase activity in Eif2ak4–/– mice with genetic disruption of the kinase domain induced neither PVOD nor pulmonary hypertension (PH) but inhibited hypoxia-induced PH. RNA-sequencing analysis suggested endothelin-1 (Edn1) as a downstream target of GCN2. GCN2 mediated hypoxia-induced Edn1 expression in human lung ECs via HIF-2α. Restored Edn1 expression in ECs of Eif2ak4–/– mice partially reversed the reduced phenotype of hypoxia-induced PH. Furthermore, GCN2 kinase inhibitor A-92 treatment attenuated PAH in monocrotaline-treated rats. These studies demonstrate that GCN2 kinase activation mediates pulmonary vascular remodeling and PAH at least partially through Edn1. Thus, targeting GCN2 kinase activation is a promising therapeutic strategy for treatment of PAH in patients without EIF2AK4 loss-of-function mutations.
Authors
Maggie M. Zhu, Jingbo Dai, Zhiyu Dai, Yi Peng, You-Yang Zhao
×Abstract
Many circulating proteins are associated with risk of ESKD, but their source and the biological pathways/disease processes they represent are unclear. Using OLINK proteomics platform, concentrations of 455 proteins were measured in plasma specimens obtained at baseline from 399 individuals with diabetes. Elevated concentrations of 46 circulating proteins were associated (P < 1 × 10–5) with development of ESKD (n = 143) during 7–15 years of follow-up. Twenty of these proteins enriched apoptosis/TNF receptor signaling pathways. A subset of 20 proteins (5–7 proteins), summarized as an apoptosis score, together with clinical variables accurately predicted risk of ESKD. Expression of genes encoding the 46 proteins in peripheral WBCs showed no difference between cells from individuals who did or did not develop ESKD. In contrast, plasma concentration of many of the 46 proteins differed by this outcome. In single-nucleus RNA-Seq analysis of kidney biopsies, the majority of genes encoding for the 20 apoptosis/TNF receptor proteins were overexpressed in injured versus healthy proximal tubule cells. Expression of these 20 genes also correlated with the overall index of apoptosis in these cells. Elevated levels of circulating proteins flagging apoptotic processes/TNF receptor signaling pathways — and likely originating from kidney cells, including injured/apoptotic proximal tubular cells — preceded the development of ESKD.
Authors
Katsuhito Ihara, Eiichiro Satake, Parker C. Wilson, Bozena Krolewski, Hiroki Kobayashi, Zaipul I. Md Dom, Joseph Ricca, Jonathan Wilson, Jonathan M. Dreyfuss, Monika A. Niewczas, Alessandro Doria, Robert G. Nelson, Marcus G. Pezzolesi, Benjamin D. Humphreys, Kevin Duffin, Andrzej S. Krolewski
×Abstract
BACKGROUND Mitochondrial diseases belong to the group of inborn errors of metabolism (IEM), with a prevalence of 1 in 2,000–5,000 individuals. They are the most common form of IEM, but, despite advances in next-generation sequencing technologies, almost half of the patients are left genetically undiagnosed.METHODS We investigated a cohort of 61 patients with defined mitochondrial disease to improve diagnostics, identify biomarkers, and correlate metabolic pathways to specific disease groups. Clinical presentations were structured using human phenotype ontology terms, and mass spectrometry–based proteomics was performed on primary fibroblasts. Additionally, we integrated 6 patients carrying variants of uncertain significance (VUS) to test proteomics as a diagnostic expansion.RESULTS Proteomic profiles from patient samples could be classified according to their biochemical and genetic characteristics, with the expression of 5 proteins (GPX4, MORF4L1, MOXD1, MSRA, and TMED9) correlating with the disease cohort, thus acting as putative biomarkers. Pathway analysis showed a deregulation of inflammatory and mitochondrial stress responses. This included the upregulation of glycosphingolipid metabolism and mitochondrial protein import, as well as the downregulation of arachidonic acid metabolism. Furthermore, we could assign pathogenicity to a VUS in MRPS23 by demonstrating the loss of associated mitochondrial ribosome subunits.CONCLUSION We established mass spectrometry–based proteomics on patient fibroblasts as a viable and versatile tool for diagnosing patients with mitochondrial disease.FUNDING The NovoNordisk Foundation, Knut and Alice Wallenberg Foundation, Wellcome Centre for Mitochondrial Research, UK Medical Research Council, and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children.
Authors
Sandrina P. Correia, Marco F. Moedas, Lucie S. Taylor, Karin Naess, Albert Z. Lim, Robert McFarland, Zuzanna Kazior, Anastasia Rumyantseva, Rolf Wibom, Martin Engvall, Helene Bruhn, Nicole Lesko, Ákos Végvári, Lukas Käll, Matthias Trost, Charlotte L. Alston, Christoph Freyer, Robert W. Taylor, Anna Wedell, Anna Wredenberg
×Abstract
Macrophage transition from an inflammatory to reparative phenotype after tissue injury is controlled by epigenetic enzymes that regulate inflammatory gene expression. We have previously identified that the histone methyltransferase SETDB2 in macrophages drives tissue repair by repressing NF-κB–mediated inflammation. Complementary ATAC-Seq and RNA-Seq of wound macrophages isolated from mice deficient in SETDB2 in myeloid cells revealed that SETDB2 suppresses the inflammatory gene program by inhibiting chromatin accessibility at NF-κB–dependent gene promoters. We found that STAT3 was required for SETDB2 expression in macrophages, yet paradoxically, it also functioned as a binding partner of SETDB2 where it repressed SETDB2 activity by inhibiting its interaction with the NF-κB component, RELA, leading to increased RELA/NF-κB–mediated inflammatory gene expression. Furthermore, RNA-Seq in wound macrophages from STAT3-deficient mice corroborated this and revealed STAT3 and SETDB2 transcriptionally coregulate overlapping genes. Finally, in diabetic wound macrophages, STAT3 expression and STAT3/SETDB2 binding were increased. We have identified what we believe to be a novel STAT3/SETDB2 axis that modulates macrophage phenotype during tissue repair and may be an important therapeutic target for nonhealing diabetic wounds.
Authors
Kevin D. Mangum, Aaron denDekker, Qinmengge Li, Lam C. Tsoi, Amrita D. Joshi, William J. Melvin, Sonya J. Wolf, Jadie Y. Moon, Christopher O. Audu, James Shadiow, Andrea T. Obi, Rachael Wasikowski, Emily C. Barrett, Tyler M. Bauer, Kylie Boyer, Zara Ahmed, Frank M. Davis, Johann Gudjonsson, Katherine A. Gallagher
×Abstract
Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step toward the development of disease-modifying therapies.
Authors
Ruth D. Azaria, Adele B. Correia, Kylie J. Schache, Manuela Zapata, Koralege C. Pathmasiri, Varshasnata Mohanty, Dharma T. Nannapaneni, Brandon L. Ashfeld, Paul Helquist, Olaf Wiest, Kenji Ohgane, Qingqing Li, Ross A. Fredenburg, Brian S.J. Blagg, Stephanie M. Cologna, Mark L. Schultz, Andrew P. Lieberman
×Abstract
Lichen planus (LP) is a chronic, debilitating, inflammatory disease of the skin and mucous membranes that affects 1%–2% of Americans. Its molecular pathogenesis remains poorly understood, and there are no FDA-approved treatments. We performed single-cell RNA sequencing on paired blood and skin samples (lesional and nonlesional tissue) from 7 patients with LP. We discovered that LP keratinocytes and fibroblasts specifically secrete a combination of CXCL9, CXCL10, and CCL19 cytokines. Using an in vitro migration assay with primary human T cells, we demonstrated that CCL19 in combination with either of the other 2 cytokines synergistically enhanced recruitment of CD8+ T cells more than any individual cytokine. Moreover, exhausted T cells in lesional LP skin secreted CXCL13, which, along with CCL19, also enhanced recruitment of T cells, suggesting a feed-forward loop in LP. Finally, LP blood revealed decreased circulating naive CD8+ T cells compared with that in healthy volunteers, consistent with recruitment to skin. Molecular analysis of LP skin and blood samples increased our understanding of disease pathogenesis and identified CCL19 as a new therapeutic target for treatment.
Authors
Anna E. Kersh, Satish Sati, Jianhe Huang, Christina Murphy, Olivia Ahart, Thomas H. Leung
×Abstract
Spinal muscular atrophy (SMA) is a recessive developmental disorder caused by the genetic loss or mutation of the gene SMN1 (survival of motor neuron 1). SMA is characterized by neuromuscular symptoms and muscle weakness. Several years ago, SMA treatment underwent a radical transformation, with the approval of 3 different SMN-dependent disease-modifying therapies. This includes 2 SMN2 splicing therapies — risdiplam and nusinersen. One main challenge for type II SMA patients treated with these drugs is ongoing muscle fatigue, limited mobility, and other skeletal problems. To date, few molecular studies have been conducted on SMA patient–derived tissues after treatment, limiting our understanding of what targets remain unchanged after the spinal cord–targeted therapies are applied. Therefore, we collected paravertebral muscle from 8 type II patients undergoing spinal surgery for scoliosis and 7 controls. We used RNA-seq to characterize their transcriptional profiles and correlate these molecular changes with muscle histology. Despite the limited cohort size and heterogeneity, we observed a consistent loss of oxidative phosphorylation (OXPHOS) machinery of the mitochondria, a decrease in mitochondrial DNA copy number, and a correlation between signals of cellular stress, denervation, and increased fibrosis. This work provides new putative targets for combination therapies for type II SMA.
Authors
Fiorella Carla Grandi, Stéphanie Astord, Sonia Pezet, Elèna Gidaja, Sabrina Mazzucchi, Maud Chapart, Stéphane Vasseur, Kamel Mamchaoui, Piera Smeriglio
×Abstract
SCN8A developmental and epileptic encephalopathy (DEE) is a severe epilepsy syndrome resulting from mutations in the voltage-gated sodium channel Nav1.6, encoded by the gene SCN8A. Nav1.6 is expressed in excitatory and inhibitory neurons, yet previous studies primarily focus on how SCN8A mutations affect excitatory neurons, with limited studies on the importance of inhibitory interneurons. Parvalbumin (PV) interneurons are a prominent inhibitory interneuron subtype that expresses Nav1.6. To assess PV interneuron function within SCN8A DEE, we used 2 mouse models harboring patient-derived SCN8A gain-of-function variants, Scn8aD/+, where the SCN8A variant N1768D is expressed globally, and Scn8aW/+-PV, where the SCN8A variant R1872W is selectively expressed in PV interneurons. Expression of the R1872W SCN8A variant selectively in PV interneurons led to development of spontaneous seizures and seizure-induced death. Electrophysiology studies showed that Scn8aD/+ and Scn8aW/+-PV interneurons were susceptible to depolarization block and exhibited increased persistent sodium current. Evaluation of synaptic connections between PV interneurons and pyramidal cells showed synaptic transmission deficits in Scn8aD/+ and Scn8aW/+-PV interneurons. Together, our findings indicate that PV interneuron failure via depolarization block along with inhibitory synaptic impairment likely elicits an overall inhibitory reduction in SCN8A DEE, leading to unchecked excitation and ultimately resulting in seizures and seizure-induced death.
Authors
Raquel M. Miralles, Alexis R. Boscia, Shrinidhi Kittur, Jessica C. Hanflink, Payal S. Panchal, Matthew S. Yorek, Tyler C. J. Deutsch, Caeley M. Reever, Shreya R. Vundela, Eric R. Wengert, Manoj K. Patel
×Abstract
With the increasing prevalence of antimicrobial-resistant bacterial infections, there is interest in using bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of i.v. administered bacteriophage in uninfected mice. A single dose of LPS-5, a bacteriophage recently used in human clinical trials to treat drug-resistant Pseudomonas aeruginosa, was administered i.v. to both immunocompetent BALB/c and neutropenic CD1 mice. Phage concentrations were assessed in peripheral blood and spleen at 0.25, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance was only minimally affected by neutropenia. Indeed, the half-lives of phages in blood in BALB/c and CD1 mice were 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that significant inactivation of circulating phages occurs over time. These data indicate that alternative factors, but not neutrophils, inactivate i.v. administered phages.
Authors
Arne Echterhof, Tejas Dharmaraj, Arya Khosravi, Robert McBride, Lynn Miesel, Ju-Hsin Chia, Patrick M. Blankenberg, Kun-Yuan Lin, Chien-Chang Shen, Yu-Ling Lee, Yu-Chuan Yeh, Wei Ting Liao, Francis G. Blankenberg, Krystyna Dąbrowska, Derek F. Amanatullah, Adam R. Frymoyer, Paul L. Bollyky
×Abstract
Breastfeeding provides important immunological benefits to the neonate, but how the different immunoactive components in breastmilk contribute to immunity remains poorly understood. Here, we characterized human breastmilk T cells using single-cell RNA-Seq and flow cytometry. Breastmilk contained predominantly memory T cells, with expression of immune signaling genes, high proliferation, and an effector Th1/cytotoxic profile with high cytokine production capacities. Elevated activation was balanced by an enriched Treg population and immune regulatory markers in conventional memory T cells. Gene and surface expression of tissue-residency markers indicate that breastmilk T cells represented tissue-adapted rather than circulatory T cells. In addition, breastmilk T cells had a broad homing profile and higher activation markers in these migratory subsets. The partly overlapping transcriptome profile between breastmilk and breast tissue T cells, particularly cytotoxic T cells, might support a role in local immune defense in the mammary gland. However, unique features of breastmilk, such as Tregs, might imply an additional role in neonatal immune support. We found some correlations between the breastmilk T cell profile and clinical parameters, most notably with maternal and household factors. Together, our data suggest that breastmilk contains an adapted T cell population that exerts their function in specific tissue sites.
Authors
Elise S. Saager, Arthur H. van Stigt, Butstabong Lerkvaleekul, Lisanne Lutter, Anneke H. Hellinga, M. Marlot van der Wal, Louis J. Bont, Jeanette H.W. Leusen, Belinda van’t Land, Femke van Wijk, the Protection against Respiratory tract infections through human Milk Analysis (PRIMA) group
×Abstract
After transplantation, Th1-mediated immune rejection is the predominant cause of graft failure. Th1 cell sensitization occurs through complex and context-dependent interaction among antigen-presenting cell subsets, particularly CD11b+ DCs (DC2) and CD103+ DCs (DC1). This interaction necessitates further investigation in the context of transplant immunity. We used well-established preclinical models of corneal transplantation and identified distinct roles of migratory CD103+ DC1 in influencing the outcomes of the grafted tissue. In recipients with uninflamed corneal beds, migratory CD103+ DC1 demonstrate a tolerogenic phenotype that modulates the immunogenic capacity of CD11b+ DC2 primarily mediated by IL-10, suppressing alloreactive CD4+ Th1 cells via the PD-L1/PD-1 pathway and enhancing Treg-mediated tolerance via αvβ8 integrin–activated TGF-β1, thus facilitating graft survival. Conversely, in recipients with inflamed and vascularized corneal beds, IFN-γ produced by CD4+ Th1 cells induced migratory CD103+ DC1 to adopt an immunostimulatory phenotype, characterized by the downregulation of regulatory markers, including αvβ8 integrin and IL-10, and the upregulation of IL-12 and costimulatory molecules CD80/86, resulting in graft failure. The adoptive transfer of ex vivo induced tolerogenic CD103+ DC1 (iDC1) effectively inhibited Th1 polarization and preserved the tolerogenic phenotype of their physiological counterparts. Collectively, our findings underscore the essential role played by CD103+ DC1 in modulating host alloimmune responses.
Authors
Tomás Blanco, Hayate Nakagawa, Aytan Musayeva, Mark Krauthammer, Rohan Bir Singh, Akitomo Narimatsu, Hongyan Ge, Sara I. Shoushtari, Reza Dana
×Abstract
Tumor-associated neutrophils (TANs) have been shown to promote immunosuppression and tumor progression, and a high TAN frequency predicts poor prognosis in triple-negative breast cancer (TNBC). Dysregulation of CREB-binding protein (CBP)/P300 function has been observed with multiple cancer types. The bromodomain (BRD) of CBP/P300 has been shown to regulate its activity. In this study, we found that IACS-70654, a selective CBP/P300 BRD inhibitor, reduced TANs and inhibited the growth of neutrophil-enriched TNBC models. In the bone marrow, CBP/P300 BRD inhibition reduced the tumor-driven abnormal differentiation and proliferation of neutrophil progenitors. Inhibition of CBP/P300 BRD also stimulated the immune response by inducing an IFN response and MHCI expression in tumor cells and increasing tumor-infiltrated cytotoxic T cells. Moreover, IACS-70654 improved the response of a neutrophil-enriched TNBC model to docetaxel and immune checkpoint blockade. This provides a rationale for combining a CBP/P300 BRD inhibitor with standard-of-care therapies in future clinical trials for neutrophil-enriched TNBC.
Authors
Xueying Yuan, Xiaoxin Hao, Hilda L. Chan, Na Zhao, Diego A. Pedroza, Fengshuo Liu, Kang Le, Alex J. Smith, Sebastian J. Calderon, Nadia Lieu, Michael J. Soth, Philip Jones, Xiang H.F. Zhang, Jeffrey M. Rosen
×Abstract
Elite controllers (ECs), a unique group of people with HIV (PWH), exhibit remarkable control of viral replication in the absence of antiretroviral therapy. In this study, we comprehensively characterized the NK cell repertoire in ECs after long-term viral control. Phenotypic profiling of NK cells revealed profound differences compared with other PWH, but marked similarities to uninfected individuals, with a distinctive prevalence of NKG2C+CD57+ memory-like NK cells. Functional analyses indicated that ECs had limited production of functional molecules upon NK stimulation and consequently reduced natural cytotoxicity against non-HIV target cells. Importantly, ECs showed an exceptional ability to kill primary HIV-infected cells by the antibody-dependent cell cytotoxicity adaptive mechanism, which was achieved by a specific memory-like NK population expressing CD16, NKG2A, NKG2C, CD57, and CXCR3. In-depth single-cell RNA-seq unveiled a unique transcriptional signature in these NK cells linked to increased cell metabolism, migration, chemotaxis, effector functions, cytokine secretion, and antiviral response. Our findings underscore a pivotal role of NK cells in the immune control of HIV and identify specific NK cells as emerging targets for immunotherapies.
Authors
Nerea Sánchez-Gaona, Ana Gallego-Cortés, Antonio Astorga-Gamaza, Norma Rallón, José Miguel Benito, Ezequiel Ruiz-Mateos, Adrian Curran, Joaquin Burgos, Jordi Navarro, Paula Suanzes, Vicenç Falcó, Meritxell Genescà, Maria J. Buzon
×Abstract
BACKGROUND Prostate cancer (PC) is driven by aberrant signaling of the androgen receptor (AR) or its ligands, and androgen deprivation therapies (ADTs) are a cornerstone of treatment. ADT responsiveness may be associated with germline changes in genes that regulate androgen production, uptake, and conversion (APUC).METHODS We analyzed whole-exome sequencing (WES) and whole-transcriptome sequencing (WTS) data from prostate tissues (SU2C/PCF, TCGA, GETx). We also interrogated the Caris Precision Oncology Alliance (POA) DNA (592-gene/whole exome) and RNA (whole transcriptome) next-generation sequencing databases. Algorithm for Linking Activity Networks (ALAN) was used to quantify all pairwise gene-to-gene associations. Real-world overall survival was determined from insurance claims data using Kaplan-Meier estimates.RESULTS Six APUC genes (HSD3B1, HSD3B2, CYP3A43, CYP11A1, CYP11B1, CYP17A1) exhibited coalescent gene behavior in a cohort of metastatic tumors (n = 208). In the Caris POA dataset, the 6 APUC genes (APUC-6) exhibited robust clustering in primary prostate (n = 4,490) and metastatic (n = 2,593) biopsies. Surprisingly, tumors with elevated APUC-6 expression had statically lower expression of AR, AR-V7, and AR signaling scores, suggesting ligand-driven disease biology. APUC-6 genes instead associated with the expression of alternative steroid hormone receptors, ESR1/2 and PGR. We used RNA expression of AR or APUC-6 genes to define 2 subgroups of tumors with differential association with hallmark pathways and cell surface targets.CONCLUSIONS The APUC-6-high/AR-low tumors represented a subgroup of patients with good clinical outcomes, in contrast with the AR-high or neuroendocrine PCs. Altogether, measuring the aggregate expression of APUC-6 genes in current genomic tests identifies PCs that are ligand (rather than AR) driven and require distinct therapeutic strategies.FUNDING NCI/NIH 1R37CA288972-01, NCI Cancer Center Support P30 CA077598, DOD W81XWH-22-2-0025, R01 CA249279.
Authors
Hannah E. Bergom, Ella Boytim, Sean McSweeney, Negar Sadeghipour, Andrew Elliott, Rachel Passow, Eamon Toye, Xiuxiu Li, Pornlada Likasitwatanakul, Daniel M. Geynisman, Scott M. Dehm, Susan Halabi, Nima Sharifi, Emmanuel S. Antonarakis, Charles J. Ryan, Justin Hwang
×Abstract
Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and long COVID are debilitating multisystemic conditions sharing similarities in immune dysregulation and cellular signaling pathways contributing to the pathophysiology. In this study, immune exhaustion gene expression was investigated in participants with ME/CFS or long COVID concurrently. RNA was extracted from peripheral blood mononuclear cells isolated from participants with ME/CFS (n = 14), participants with long COVID (n = 15), and healthy controls (n = 18). Participants with ME/CFS were included according to Canadian Consensus Criteria. Participants with long COVID were eligible according to the case definition for “Post COVID-19 Condition” published by the World Health Organization. RNA was analyzed using the NanoString nCounter Immune Exhaustion gene expression panel. Differential gene expression analysis in ME/CFS revealed downregulated IFN signaling and immunoglobulin genes, and this suggested a state of immune suppression. Pathway analysis implicated dysregulated macrophage activation, cytokine production, and immunodeficiency signaling. Long COVID samples exhibited dysregulated expression of genes regarding antigen presentation, cytokine signaling, and immune activation. Differentially expressed genes were associated with antigen presentation, B cell development, macrophage activation, and cytokine signaling. This investigation elucidates the intricate role of both adaptive and innate immune dysregulation underlying ME/CFS and long COVID, emphasizing the potential importance of immune exhaustion in disease progression.
Authors
Natalie Eaton-Fitch, Penny Rudd, Teagan Er, Livia Hool, Lara Herrero, Sonya Marshall-Gradisnik
×Abstract
Despite advances in sequencing technologies, a molecular diagnosis remains elusive in many patients with Mendelian disease. Current short-read clinical sequencing approaches cannot provide chromosomal phase information or epigenetic information without further sample processing, which is not routinely done and can result in an incomplete molecular diagnosis in patients. The ability to provide phased genetic and epigenetic information from a single sequencing run would improve the diagnostic rate of Mendelian conditions. Here, we describe targeted long-read sequencing of Mendelian disease genes (TaLon-SeqMD) using a real-time adaptive sequencing approach. Optimization of bioinformatic targeting enabled selective enrichment of multiple disease-causing regions of the human genome. Haplotype-resolved variant calling and simultaneous resolution of epigenetic base modification could be achieved in a single sequencing run. The TaLon-SeqMD approach was validated in a cohort of 18 individuals with previous genetic testing targeting 373 inherited retinal disease (IRD) genes, yielding the complete molecular diagnosis in each case. This approach was then applied in 2 IRD cases with inconclusive testing, which uncovered noncoding and structural variants that were difficult to characterize by standard short-read sequencing. Overall, these results demonstrate TaLon-SeqMD as an approach to provide rapid phased-variant calling to provide the molecular basis of Mendelian diseases.
Authors
Kenji Nakamichi, Jennifer Huey, Riccardo Sangermano, Emily M. Place, Kinga M. Bujakowska, Molly Marra, Lesley A. Everett, Paul Yang, Jennifer R. Chao, Russell N. Van Gelder, Debarshi Mustafi
×Abstract
BACKGROUND Despite the currently prevailing, milder Omicron variant of COVID-19, older adults remain at elevated risk of hospital admission, critical illness, and death. Loss of efficacy of the immune system, including reduced strength, quality, and durability of antibody responses, may render generalized recommendations on booster vaccinations inadequate. There is a lack of data on the efficacy of antibody levels in older adults and on the utility of vaccination status versus antibody levels as a correlate of protection. It is further unclear whether antibody levels may be used to guide the timing of booster vaccinations in older adults.METHODS We conducted a prospective multicenter cohort study comprising hospitalized patients with COVID-19. Anti–SARS-CoV-2 spike antibodies were measured on hospital admission. The primary endpoint was in-hospital mortality. Patients were stratified by age, antibody levels, and vaccination status. Multiple logistic regression and Cox regression analyses were conducted.RESULTS In total, 785 older patients (≥60 years of age [a]) and 367 controls (<60a) were included. After adjusting for confounders, risk of mortality, ICU admission, endotracheal intubation, and oxygen administration was 4.9, 2.6, 6.5, and 2.3 times higher, respectively, if antibody levels were < 1,200 BAU/mL (aOR, 4.92 [95%CI, 2.59–9.34], P < 0.0001; aOR, 2.64 [95%CI, 1.52–4.62], P = 0.0006; aOR, 6.50 [95%CI, 1.48–28.47], P = 0.013; aOR, 2.34 [95%CI, 1.60–3.343], P < 0.0001). Older adults infected with the Omicron variant were approximately 6 times more likely to die if antibody levels were < 1,200 BAU/mL (aOR, 6.3 [95% CI, 2.43–16.40], P = 0.0002).CONCLUSION Antibody levels were a stronger predictor of in-hospital mortality than vaccination status. Monitoring antibody levels may constitute a better and more direct approach for safeguarding older adults from adverse COVID-19 outcomes.
Authors
Sylvia Mink, Christoph H. Saely, Andreas Leiherer, Patrick Reimann, Matthias Frick, Janne Cadamuro, Wolfgang Hitzl, Heinz Drexel, Peter Fraunberger
×Abstract
BACKGROUND Immune processes are influenced by circadian rhythms. We evaluate the association between varicella vaccine administration time of day and vaccine effectiveness.METHODS A national cohort, children younger than 6 years, were enrolled between January 2002 and December 2023. We compared children vaccinated during morning (7:00–10:59), late morning to afternoon (11:00–15:59), or evening hours (16:00–19:59). A Cox proportional hazards regression model was used to adjust for ethnicity, sex, and comorbidities. The first varicella infection occurring at least 14 days after vaccination and a second dose administration were treated as terminal events.RESULTS Of 251,141 vaccinated children, 4,501 (1.8%) experienced breakthrough infections. Infection rates differed based on vaccination time, with the lowest rates associated with late morning to afternoon (11:00–15:59), HR 0.88, 95% CI 0.82–0.95, P < 0.001, and the highest rates with evening vaccination (16:00–19:59), HR 1.41, 95% CI 1.32–1.52, P < 0.001. Vaccination timing remained significant after adjustment for ethnicity, sex, and comorbidities. The association between immunization time and infection risk followed a sinusoidal pattern, consistent with a diurnal rhythm in vaccine effectiveness.CONCLUSION We report a significant association between the time of varicella vaccination and its clinical effectiveness. Similar association was observed with the COVID-19 vaccine, providing proof of concept consistent with a diurnal rhythm in vaccine effectiveness.
Authors
Dana Danino, Yoav Kalron, Jeffrey A. Haspel, Guy Hazan
×Abstract
Lipoprotein lipase (LPL) and multiple regulators of LPL activity (e.g., APOC2 and ANGPTL4) are present in all vertebrates, but GPIHBP1—the endothelial cell (EC) protein that captures LPL within the subendothelial spaces and transports it to its site of action in the capillary lumen—is present in mammals but in not chickens or other lower vertebrates. In mammals, GPIHBP1 deficiency causes severe hypertriglyceridemia, but chickens maintain low triglyceride levels despite the absence of GPIHBP1. To understand intravascular lipolysis in lower vertebrates, we examined LPL expression in mouse and chicken hearts. In both species, LPL was abundant on capillaries, but the distribution of Lpl transcripts was strikingly different. In mouse hearts, Lpl transcripts were extremely abundant in cardiomyocytes but were barely detectable in capillary ECs. In chicken hearts, Lpl transcripts were absent in cardiomyocytes but abundant in capillary ECs. In zebrafish hearts, lpl transcripts were also in capillary ECs but not cardiomyocytes. In both mouse and chicken hearts, LPL was present, as judged by immunogold electron microscopy, in the glycocalyx of capillary ECs. Thus, mammals produce LPL in cardiomyocytes and rely on GPIHBP1 to transport the LPL into capillaries, whereas lower vertebrates produce LPL directly in capillary ECs, rendering an LPL transporter unnecessary.
Authors
Le Phuong Nguyen, Wenxin Song, Ye Yang, Anh P. Tran, Thomas A. Weston, Hyesoo Jung, Yiping Tu, Paul H. Kim, Joonyoung R. Kim, Katherine Xie, Rachel G. Yu, Julia Scheithauer, Ashley M. Presnell, Michael Ploug, Gabriel Birrane, Hannah Arnold, Katarzyna Koltowska, Maarja A. Mäe, Christer Betsholtz, Liqun He, Jeffrey L. Goodwin, Anne P. Beigneux, Loren G. Fong, Stephen G. Young
