Uric acid formation is driven by crosstalk between skeletal muscle and other cell types
Miller et al. report that, during atrophic conditions, skeletal muscle degrades endogenous purines that are released and converted to uric acid by other cell types, such as endothelial cells. The cover shows an immunofluorescence image of C2C12 myotubes, which are differentiated mouse muscle cells, displaying myosin heavy chain (orange) and nuclei (blue). Image credit: Spencer Miller.
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Technical Advance
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Abstract
With antimicrobial resistance (AMR) emerging as a major threat to global health, monoclonal antibodies (MAbs) have become a promising means to combat difficult-to-treat AMR infections. Unfortunately, in contrast with standard antimicrobials, for which there are well-validated clinical laboratory methodologies to determine whether an infecting pathogen is susceptible or resistant to a specific antimicrobial drug, no assays have been described that can inform clinical investigators or clinicians regarding the clinical efficacy of a MAb against a specific pathogenic strain. Using Acinetobacter baumannii as a model organism, we established and validated 2 facile clinical susceptibility assays, which used flow cytometry and latex bead agglutination, to determine susceptibility (predicting in vivo efficacy) or resistance (predicting in vivo failure) of 1 newly established and 3 previously described anti–A. baumannii MAbs. These simple assays exhibited impressive sensitivity, specificity, and reproducibility, with clear susceptibility breakpoints that predicted the in vivo outcomes in our preclinical model with excellent fidelity. These MAb susceptibility assays have the potential to enable and facilitate clinical development and deployment of MAbs that generally target the surface of microbes.
Authors
Matthew J. Slarve, Neven Bowler, Elizabeth Burk, Jun Yan, Ulrike Carlino-MacDonald, Thomas A. Russo, Brian M. Luna, Brad Spellberg
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Research Articles
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Abstract
Circadian rhythm dysfunction is a hallmark of Parkinson disease (PD), and diminished expression of the core clock gene Bmal1 has been described in patients with PD. BMAL1 is required for core circadian clock function but also serves nonrhythmic functions. Germline Bmal1 deletion can cause brain oxidative stress and synapse loss in mice, and it can exacerbate dopaminergic neurodegeneration in response to the toxin MPTP. Here we examined the effect of cell type–specific Bmal1 deletion on dopaminergic neuron viability in vivo. We observed that global, postnatal deletion of Bmal1 caused spontaneous loss of tyrosine hydroxylase+ (TH+) dopaminergic neurons in the substantia nigra pars compacta (SNpc). This was not replicated by light-induced disruption of behavioral circadian rhythms and was not induced by astrocyte- or microglia-specific Bmal1 deletion. However, either pan-neuronal or TH neuron–specific Bmal1 deletion caused cell-autonomous loss of TH+ neurons in the SNpc. Bmal1 deletion did not change the percentage of TH neuron loss after α-synuclein fibril injection, though Bmal1-KO mice had fewer TH neurons at baseline. Transcriptomics analysis revealed dysregulation of pathways involved in oxidative phosphorylation and Parkinson disease. These findings demonstrate a cell-autonomous role for BMAL1 in regulating dopaminergic neuronal survival and may have important implications for neuroprotection in PD.
Authors
Michael F. Kanan, Patrick W. Sheehan, Jessica N. Haines, Pedro G. Gomez, Adya Dhuler, Collin J. Nadarajah, Zachary M. Wargel, Brittany M. Freeberg, Hemanth R. Nelvagal, Mariko Izumo, Joseph S. Takahashi, Jonathan D. Cooper, Albert A. Davis, Erik S. Musiek
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The resting zone of the postnatal growth plate is organized by slow-cycling chondrocytes expressing parathyroid hormone-related protein (PTHrP), which include a subgroup of skeletal stem cells that contribute to the formation of columnar chondrocytes. The PTHrP–Indian hedgehog feedback regulation is essential for sustaining growth plate activities; however, molecular mechanisms regulating cell fates of PTHrP+ resting chondrocytes and their eventual transformation into osteoblasts remain largely undefined. Here, in a mouse model, we specifically activated Hedgehog signaling in PTHrP+ resting chondrocytes and traced the fate of their descendants using a tamoxifen-inducible Pthrp-creER line with patched-1–floxed and tdTomato reporter alleles. Hedgehog-activated PTHrP+ chondrocytes formed large, concentric, clonally expanded cell populations within the resting zone (“patched roses”) and generated significantly wider columns of chondrocytes, resulting in hyperplasia of the growth plate. Interestingly, Hedgehog-activated PTHrP+ cell descendants migrated away from the growth plate and transformed into trabecular osteoblasts in the diaphyseal marrow space in the long term. Therefore, Hedgehog activation drives resting zone chondrocytes into transit-amplifying states as proliferating chondrocytes and eventually converts these cells into osteoblasts, unraveling a potentially novel Hedgehog-mediated mechanism that facilitates osteogenic cell fates of PTHrP+ skeletal stem cells.
Authors
Shion Orikasa, Yuki Matsushita, Hiroaki Manabe, Michael Fogge, Zachary Lee, Koji Mizuhashi, Naoko Sakagami, Wanida Ono, Noriaki Ono
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Infection of immature mice with rhinovirus (RV) induces an asthma-like phenotype consisting of type 2 inflammation, mucous metaplasia, eosinophilic inflammation, and airway hyperresponsiveness that is dependent on IL-25 and type 2 innate lymphoid cells (ILC2s). Doublecortin-like kinase 1–positive (DCLK1+) tuft cells are a major source of IL-25. We sought to determine the requirement of tuft cells for the RV-induced asthma phenotype in wild-type mice and mice deficient in Pou2f3, a transcription factor required for tuft cell development. C57BL/6J mice infected with RV-A1B on day 6 of life and RV-A2 on day 13 of life showed increased DCLK1+ tuft cells in the large airways. Compared with wild-type mice, RV-infected Pou2f3–/– mice showed reductions in IL-25 mRNA and protein expression, ILC2 expansion, type 2 cytokine expression, mucous metaplasia, lung eosinophils, and airway methacholine responsiveness. We conclude that airway tuft cells are required for the asthma phenotype observed in immature mice undergoing repeated RV infections. Furthermore, RV-induced tuft cell development provides a mechanism by which early-life viral infections could potentiate type 2 inflammatory responses to future infections.
Authors
Yiran Li, Mingyuan Han, Shilpi Singh, Haley A. Breckenridge, Jordan E. Kreger, Claudia C. Stroupe, Daniel A. Sawicky, Shiuhyang Kuo, Adam M. Goldsmith, Fang Ke, Anukul T. Shenoy, J. Kelley Bentley, Ichiro Matsumoto, Marc B. Hershenson
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Cigarette smoking is associated with a higher risk of ICU admissions among patients with flu. However, the etiological mechanism by which cigarette smoke (CS) exacerbates flu remains poorly understood. Here, we show that a mild dose of influenza A virus promotes a severe lung injury in mice preexposed to CS but not room air for 4 weeks. Real-time intravital (in vivo) lung imaging revealed that the development of acute severe respiratory dysfunction in CS- and flu-exposed mice was associated with the accumulation of platelet-rich neutrophil-platelet aggregates (NPAs) in the lung microcirculation within 2 days following flu infection. These platelet-rich NPAs formed in situ and grew larger over time to occlude the lung microvasculature, leading to the development of pulmonary ischemia followed by the infiltration of NPAs and vascular leakage into the alveolar air space. These findings suggest, for the first time to our knowledge, that an acute onset of platelet-driven thrombo-inflammatory response in the lung contributes to the development of CS-induced severe flu.
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Tomasz W. Kaminski, Tomasz Brzoska, Xiuying Li, Ravi Vats, Omika Katoch, Rikesh K. Dubey, Kamal Bagale, Simon C. Watkins, Bryan J. McVerry, Tirthadipa Pradhan-Sundd, Lianghui Zhang, Keven M. Robinson, Toru Nyunoya, Prithu Sundd
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Cachexia is a debilitating skeletal muscle wasting condition for which we currently lack effective treatments. In the context of cancer, certain chemotherapeutics cause DNA damage and cellular senescence. Senescent cells exhibit chronic activation of the transcription factor NF-κB, a known mediator of the proinflammatory senescence-associated secretory phenotype (SASP) and skeletal muscle atrophy. Thus, targeting NF-κB represents a logical therapeutic strategy to alleviate unintended consequences of genotoxic drugs. Herein, we show that treatment with the IKK/NF-κB inhibitor SR12343 during a course of chemotherapy reduces markers of cellular senescence and the SASP in liver, skeletal muscle, and circulation and, correspondingly, attenuates features of skeletal muscle pathology. Lastly, we demonstrate that SR12343 mitigates chemotherapy-induced reductions in body weight, lean mass, fat mass, and muscle strength. These findings support senescent cells as a promising druggable target to counteract the SASP and skeletal muscle wasting in the context of chemotherapy.
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Davis A. Englund, Alyssa M. Jolliffe, Gabriel J. Hanson, Zaira Aversa, Xu Zhang, Xinyi Jiang, Thomas A. White, Lei Zhang, David G. Monroe, Paul D. Robbins, Laura J. Niedernhofer, Theodore M. Kamenecka, Sundeep Khosla, Nathan K. LeBrasseur
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Ischemia-reperfusion injury–induced (IRI-induced) acute kidney injury is accompanied by mononuclear phagocyte (MP) invasion and inflammation. However, systematic analysis of extracellular vesicle–carried (EV-carried) proteins mediating intercellular crosstalk in the IRI microenvironment is still lacking. Multiomics analysis combining single-cell RNA-Seq data of kidney and protein profiling of kidney-EV was used to elucidate the intercellular communication between proximal tubular cells (PTs) and MP. Targeted adhesion and migration of various MPs were caused by the secretion of multiple chemokines as well as integrin β1–rich EV by ischemic-damaged PTs after IRI. These recruited MPs, especially Fn1+ macrophagocyte, amplified the surviving PT’s inflammatory response by secreting the inflammatory factors TNF-α, MCP-1, and thrombospondin 1 (THBS-1), which could interact with integrin β1 to promote more MP adhesion and interact with surviving PT to further promote the secretion of IL-1β. However, GW4869 reduced MP infiltration and maintained a moderate inflammatory level likely by blocking EV secretion. Our findings establish the molecular bases by which chemokines and kidney-EV mediate PT-MP crosstalk in early IRI and provide insights into systematic intercellular communication.
Authors
Wenjuan Wang, Xuejing Ren, Xiangmei Chen, Quan Hong, Guangyan Cai
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Bile acids (BAs) affect the intestinal environment by ensuring barrier integrity, maintaining microbiota balance, regulating epithelium turnover, and modulating the immune system. As a master regulator of BA homeostasis, farnesoid X receptor (FXR) is severely compromised in patients with inflammatory bowel disease (IBD) and colitis-associated colorectal cancer (CAC). At the front line, gut macrophages react to the microbiota and metabolites that breach the epithelium. We aim to study the role of the BA/FXR axis in macrophages. This study demonstrates that inflammation-induced epithelial abnormalities compromised FXR signaling and altered BAs’ profile in a mouse CAC model. Further, gut macrophage–intrinsic FXR sensed aberrant BAs, leading to pro-inflammatory cytokines’ secretion, which promoted intestinal stem cell proliferation. Mechanistically, activation of FXR ameliorated intestinal inflammation and inhibited colitis-associated tumor growth, by regulating gut macrophages’ recruitment, polarization, and crosstalk with Th17 cells. However, deletion of FXR in bone marrow or gut macrophages escalated the intestinal inflammation. In summary, our study reveals a distinctive regulatory role of FXR in gut macrophages, suggesting its potential as a therapeutic target for addressing IBD and CAC.
Authors
Xingchen Dong, Ming Qi, Chunmiao Cai, Yu Zhu, Yuwenbin Li, Sally Coulter, Fei Sun, Christopher Liddle, Nataliya V. Uboha, Richard Halberg, Wei Xu, Paul Marker, Ting Fu
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Pain of unknown etiology is frequent in individuals with the tumor predisposition syndrome neurofibromatosis 1 (NF1), even when tumors are absent. Nerve Schwann cells (SCs) were recently shown to play roles in nociceptive processing, and we find that chemogenetic activation of SCs is sufficient to induce afferent and behavioral mechanical hypersensitivity in wild-type mice. In mouse models, animals showed afferent and behavioral hypersensitivity when SCs, but not neurons, lacked Nf1. Importantly, hypersensitivity corresponded with SC-specific upregulation of mRNA encoding glial cell line–derived neurotrophic factor (GDNF), independently of the presence of tumors. Neuropathic pain-like behaviors in the NF1 mice were inhibited by either chemogenetic silencing of SC calcium or by systemic delivery of GDNF-targeting antibodies. Together, these findings suggest that alterations in SCs directly modulate mechanical pain and suggest cell-specific treatment strategies to ameliorate pain in individuals with NF1.
Authors
Namrata G.R. Raut, Laura A. Maile, Leila M. Oswalt, Irati Mitxelena, Aaditya Adlakha, Kourtney L. Sprague, Ashley R. Rupert, Lane Bokros, Megan C. Hofmann, Jennifer Patritti-Cram, Tilat A. Rizvi, Luis F. Queme, Kwangmin Choi, Nancy Ratner, Michael P. Jankowski
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Hyperuricemia is implicated in numerous pathologies, but the mechanisms underlying uric acid production are poorly understood. Using a combination of mouse studies, cell culture studies, and human serum samples, we sought to determine the cellular source of uric acid. In mice, fasting and glucocorticoid treatment increased serum uric acid and uric acid release from ex vivo–incubated skeletal muscle. In vitro, glucocorticoids and the transcription factor FoxO3 increased purine nucleotide degradation and purine release from differentiated muscle cells, which coincided with the transcriptional upregulation of AMP deaminase 3, a rate-limiting enzyme in adenine nucleotide degradation. Heavy isotope tracing during coculture experiments revealed that oxidation of muscle purines to uric acid required their transfer from muscle cells to a cell type that expresses xanthine oxidoreductase, such as endothelial cells. Last, in healthy women, matched for age and body composition, serum uric acid was greater in individuals scoring below average on standard physical function assessments. Together, these studies reveal skeletal muscle purine degradation is an underlying driver of uric acid production, with the final step of uric acid production occurring primarily in a nonmuscle cell type. This suggests that skeletal muscle fiber purine degradation may represent a therapeutic target to reduce serum uric acid and treat numerous pathologies.
Authors
Spencer G. Miller, Catalina Matias, Paul S. Hafen, Andrew S. Law, Carol A. Witczak, Jeffrey J. Brault
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Lupus nephritis (LN) is a pathologically heterogenous autoimmune disease linked to end-stage kidney disease and mortality. Better therapeutic strategies are needed as only 30%–40% of patients completely respond to treatment. Noninvasive biomarkers of intrarenal inflammation may guide more precise approaches. Because urine collects the byproducts of kidney inflammation, we studied the urine proteomic profiles of 225 patients with LN (573 samples) in the longitudinal Accelerating Medicines Partnership in RA/SLE cohort. Urinary biomarkers of monocyte/neutrophil degranulation (i.e., PR3, S100A8, azurocidin, catalase, cathepsins, MMP8), macrophage activation (i.e., CD163, CD206, galectin-1), wound healing/matrix degradation (i.e., nidogen-1, decorin), and IL-16 characterized the aggressive proliferative LN classes and significantly correlated with histological activity. A decline of these biomarkers after 3 months of treatment predicted the 1-year response more robustly than proteinuria, the standard of care (AUC: CD206 0.91, EGFR 0.9, CD163 0.89, proteinuria 0.8). Candidate biomarkers were validated and provide potentially treatable targets. We propose these biomarkers of intrarenal immunological activity as noninvasive tools to diagnose LN and guide treatment and as surrogate endpoints for clinical trials. These findings provide insights into the processes involved in LN activity. This data set is a public resource to generate and test hypotheses and validate biomarkers.
Authors
Andrea Fava, Jill Buyon, Laurence Magder, Jeff Hodgin, Avi Rosenberg, Dawit S. Demeke, Deepak A. Rao, Arnon Arazi, Alessandra Ida Celia, Chaim Putterman, Jennifer H. Anolik, Jennifer Barnas, Maria Dall’Era, David Wofsy, Richard Furie, Diane Kamen, Kenneth Kalunian, Judith A. James, Joel Guthridge, Mohamed G. Atta, Jose Monroy Trujillo, Derek Fine, Robert Clancy, H. Michael Belmont, Peter Izmirly, William Apruzzese, Daniel Goldman, Celine C. Berthier, Paul Hoover, Nir Hacohen, Soumya Raychaudhuri, Anne Davidson, Betty Diamond, the Accelerating Medicines Partnership in RA/SLE network, Michelle Petri
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Suppression of glucagon hypersecretion can normalize hyperglycemia during type 1 diabetes (T1D). Activating erythropoietin-producing human hepatocellular receptor type-A4 (EphA4) on α cells reduced glucagon hypersecretion from dispersed α cells and T1D islets from both human donor and mouse models. We synthesized a high-affinity small molecule agonist for the EphA4 receptor, WCDD301, which showed robust plasma and liver microsome metabolic stability in both mouse and human preparations. In islets and dispersed islet cells from nondiabetic and T1D human donors, WCDD301 reduced glucagon secretion comparable to the natural EphA4 ligand, Ephrin-A5. In diabetic NOD and streptozotocin-treated mice, once-daily oral administration of WCDD301 formulated with a time-release excipient reduced plasma glucagon and normalized blood glucose for more than 3 months. These results suggest that targeting the α cell EphA4 receptor by sustained release of WCDD301 is a promising pharmacologic pathway for normalizing hyperglycemia in patients with T1D.
Authors
Farzad Asadi, Subhadra C. Gunawardana, Roland E. Dolle, David W. Piston
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Drug-induced liver injury (DILI), especially acetaminophen overdose, is the leading cause of acute liver failure. Pregnane X receptor (PXR) is a nuclear receptor and the master regulator of drug metabolism. Aberrant activation of PXR plays a pathogenic role in the acetaminophen hepatotoxicity. Here, we aimed to examine the S-nitrosylation of PXR (SNO-PXR) in response to acetaminophen. We found that PXR was S-nitrosylated in hepatocytes and the mouse livers after exposure to acetaminophen or S-nitrosoglutathione (GSNO). Mass spectrometry and site-directed mutagenesis identified the cysteine 307 as the primary residue for S-nitrosylation (SNO) modification. In hepatocytes, SNO suppressed both agonist-induced (rifampicin and SR12813) and constitutively active PXR (VP-PXR, a human PXR fused to the minimal transactivator domain of the herpes virus transcription factor VP16) activations. Furthermore, in acetaminophen-overdosed mouse livers, PXR protein was decreased at the centrilobular regions overlapping with increased SNO. In PXR–/– mice, replenishing the livers with the SNO-deficient PXR significantly aggravated hepatic necrosis, increased HMGB1 release, and exacerbated liver injury and inflammation. Particularly, we demonstrated that S-nitrosoglutathione reductase (GSNOR) inhibitor N6022 promoted hepatoprotection by increasing the levels of SNO-PXR. In conclusion, PXR is posttranslationally modified by SNO in hepatocytes in response to acetaminophen. This modification mitigated the acetaminophen-induced PXR hyperactivity. It may serve as a target for therapeutical intervention.
Authors
Qi Cui, Tingting Jiang, Xinya Xie, Haodong Wang, Lei Qian, Yanyan Cheng, Qiang Li, Tingxu Lu, Qinyu Yao, Jia Liu, Baochang Lai, Chang Chen, Lei Xiao, Nanping Wang
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There is great interest in identifying signaling pathways that promote cardiac repair after myocardial infarction (MI). Prior studies suggest a beneficial role for IL-13 signaling in neonatal heart regeneration; however, the cell types mediating cardiac regeneration and the extent of IL-13 signaling in the adult heart after injury are unknown. We identified an abundant source of IL-13 and the related cytokine, IL-4, in neonatal cardiac type 2 innate lymphoid cells, but this phenomenon declined precipitously in adult hearts. Moreover, IL-13 receptor deletion in macrophages impaired cardiac function and resulted in larger scars early after neonatal MI. By using a combination of recombinant IL-13 administration and cell-specific IL-13 receptor genetic deletion models, we found that IL-13 signaling specifically to macrophages mediated cardiac functional recovery after MI in adult mice. Single transcriptomics revealed a subpopulation of cardiac macrophages in response to IL-13 administration. These IL-13–induced macrophages were highly efferocytotic and were identified by high IL-1R2 expression. Collectively, we elucidated a strongly proreparative role for IL-13 signaling directly to macrophages following cardiac injury. While this pathway is active in proregenerative neonatal stages, reactivation of macrophage IL-13 signaling is required to promote cardiac functional recovery in adults.
Authors
Santiago Alvarez-Argote, Samantha J. Paddock, Michael A. Flinn, Caelan W. Moreno, Makenna C. Knas, Victor A. Almeida, Sydney L. Buday, Amirala Bakhshian Nik, Michaela Patterson, Yi-Guang Chen, Chien-Wei Lin, Caitlin C. O’Meara
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An arginine to cysteine substitution at amino acid position 203 (C203R) is the most common missense mutation in human cone opsin. Linked to color blindness and blue cone monochromacy (BCM), C203 is involved in a crucial disulfide bond required for proper folding. It has previously been postulated that expression of mutant C203R cone opsin exerts a toxic effect on cone photoreceptors, similar to some well-characterized missense mutations in rhodopsin that lead to protein misfolding. In this study, we generated and characterized a BCM mouse model carrying the equivalent C203R mutation (Opn1mwC198R Opn1sw–/–) to investigate the disease mechanism and develop a gene therapy approach for this disorder. Untreated Opn1mwC198R Opn1sw–/– cones phenocopied affected cones in human patients with the equivalent mutation, exhibiting shortened or absent cone outer segments and loss of function. We determined that gene augmentation targeting cones specifically yielded robust rescue of cone function and structure when Opn1mwC198R Opn1sw–/– mice were treated at early ages. Importantly, treated cones displayed elaborated outer segments and replenished expression of crucial cone phototransduction proteins. Interestingly, we were unable to detect OPN1MWC198R mutant opsin at any age. We believe this is the first proof-of-concept study exploring the efficacy of gene therapy in BCM associated with a C203R mutation.
Authors
Emily R. Sechrest, Xiaojie Ma, Marion E. Cahill, Robert J. Barbera, Yixiao Wang, Wen-Tao Deng
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The use of senolytic agents to remove senescent cells from atherosclerotic lesions is controversial. A common limitation of previous studies is the failure to rigorously define the effects of senolytic agent ABT-263 (Navitoclax) on smooth muscle cells (SMC) despite studies claiming that these cells are the major source of senescent cells. Moreover, there are no studies on the effect of ABT-263 on endothelial cells (EC), which — along with SMC — comprise 90% of α-smooth muscle actin+ (α-SMA+) myofibroblast-like cells in the protective fibrous cap. Here we tested the hypothesis that treatment of advanced atherosclerotic mice with ABT-263 will reduce lesion size and increase plaque stability. SMC (Myh11-CreERT2-eYFP) and EC (Cdh5-CreERT2-eYFP) lineage tracing Apoe–/– mice were fed a western diet (WD) for 18 weeks, followed by ABT-263 at 100 mg/kg/bw for 6 weeks or 50 mg/kg/bw for 9 weeks. ABT-263 treatment did not change lesion size or lumen area of the brachiocephalic artery (BCA). However, ABT-263 treatment reduced SMC by 90% and increased EC contributions to lesions via EC-to-mesenchymal transition (EndoMT) by 60%. ABT-263 treatment also reduced α-SMA+ fibrous cap thickness by 60% and was associated with a > 50% mortality rate. Taken together, ABT-263 treatment of WD-fed Apoe–/– mice with advanced lesions resulted in multiple detrimental changes, including reduced indices of stability and increased mortality.
Authors
Santosh Karnewar, Vaishnavi Karnewar, Laura S. Shankman, Gary K. Owens
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Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anticancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the 2 inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key enzymes PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single-agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.
Authors
Yan Zhu, Dengyong Zhang, Pooja Shukla, Young-Ho Jung, Prit Benny Malgulwar, Sharmeen Chagani, Medina Colic, Sarah Benjamin, John A. Copland III, Lin Tan, Philip L. Lorenzi, Milind Javle, Jason T. Huse, Jason Roszik, Traver Hart, Lawrence N. Kwong
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BACKGROUND Sepsis remains a major clinical challenge for which successful treatment requires greater precision in identifying patients at increased risk of adverse outcomes requiring different therapeutic approaches. Predicting clinical outcomes and immunological endotyping of septic patients generally relies on using blood protein or mRNA biomarkers, or static cell phenotyping. Here, we sought to determine whether functional immune responsiveness would yield improved precision.METHODS An ex vivo whole-blood enzyme-linked immunosorbent spot (ELISpot) assay for cellular production of interferon γ (IFN-γ) was evaluated in 107 septic and 68 nonseptic patients from 5 academic health centers using blood samples collected on days 1, 4, and 7 following ICU admission.RESULTS Compared with 46 healthy participants, unstimulated and stimulated whole-blood IFN-γ expression was either increased or unchanged, respectively, in septic and nonseptic ICU patients. However, in septic patients who did not survive 180 days, stimulated whole-blood IFN-γ expression was significantly reduced on ICU days 1, 4, and 7 (all P < 0.05), due to both significant reductions in total number of IFN-γ–producing cells and amount of IFN-γ produced per cell (all P < 0.05). Importantly, IFN-γ total expression on days 1 and 4 after admission could discriminate 180-day mortality better than absolute lymphocyte count (ALC), IL-6, and procalcitonin. Septic patients with low IFN-γ expression were older and had lower ALCs and higher soluble PD-L1 and IL-10 concentrations, consistent with an immunosuppressed endotype.CONCLUSIONS A whole-blood IFN-γ ELISpot assay can both identify septic patients at increased risk of late mortality and identify immunosuppressed septic patients.TRIAL REGISTRY N/A.FUNDING This prospective, observational, multicenter clinical study was directly supported by National Institute of General Medical Sciences grant R01 GM-139046, including a supplement (R01 GM-139046-03S1) from 2022 to 2024.
Authors
Evan L. Barrios, Monty B. Mazer, Patrick W. McGonagill, Christian B. Bergmann, Michael D. Goodman, Robert W. Gould, Mahil Rao, Valerie E. Polcz, Ruth J. Davis, Drew E. Del Toro, Marvin L.S. Dirain, Alexandra Dram, Lucas O. Hale, Mohammad Heidarian, Caleb Y. Kim, Tamara A. Kucaba, Jennifer P. Lanz, Ashley E. McCray, Sandra Meszaros, Sydney Miles, Candace R. Nelson, Ivanna L. Rocha, Elvia E. Silva, Ricardo F. Ungaro, Andrew H. Walton, Julie Xu, Leilani Zeumer-Spataro, Anne M. Drewry, Muxuan Liang, Letitia E. Bible, Tyler J. Loftus, Isaiah R. Turnbull, Philip A. Efron, Kenneth E. Remy, Scott C. Brakenridge, Vladimir P. Badovinac, Thomas S. Griffith, Lyle L. Moldawer, Richard S. Hotchkiss, Charles C. Caldwell
×Abstract
Benign prostatic hyperplasia (BPH) is the nodular proliferation of the prostate transition zone in older men, leading to urinary storage and voiding problems that can be recalcitrant to therapy. Decades ago, John McNeal proposed that BPH originates with the “reawakening” of embryonic inductive activity by adult prostate stroma, which spurs new ductal proliferation and branching morphogenesis. Here, by laser microdissection and transcriptional profiling of the BPH stroma adjacent to hyperplastic branching ducts, we identified secreted factors likely mediating stromal induction of prostate glandular epithelium and coinciding processes. The top stromal factors were insulin-like growth factor 1 (IGF1) and CXC chemokine ligand 13 (CXCL13), which we verified by RNA in situ hybridization to be coexpressed in BPH fibroblasts, along with their cognate receptors (IGF1R and CXCR5) on adjacent epithelium. In contrast, IGF1 but not CXCL13 was expressed in human embryonic prostate stroma. Finally, we demonstrated that IGF1 is necessary for the generation of BPH-1 cell spheroids and patient-derived BPH cell organoids in 3D culture. Our findings partially support historic speculations on the etiology of BPH and provide what we believe to be new molecular targets for rational therapies directed against the underlying processes driving BPH.
Authors
Anna S. Pollack, Christian A. Kunder, Noah Brazer, Zhewei Shen, Sushama Varma, Robert B. West, Gerald R. Cunha, Laurence S. Baskin, James D. Brooks, Jonathan R. Pollack
