In this issue of JCI Insight, Golden et al. evaluated the pathogenesis of SARS-CoV-2 in mice expressing the human angiotensin-converting enzyme 2 (ACE2) gene under the control of the keratin 18 promotor. They demonstrated that this model developed acute disease upon intranasal exposure, with higher levels of inflammation and mortality in male mice. The cover image shows in situ hybridization labeling of SARS-CoV-2 RNA in the olfactory epithelium of a transgenic mouse.
Lupus nephritis (LN) is a major organ complication and cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is an unmet medical need for developing more efficient and specific, mechanism-based therapies, which depends on improved understanding of the underlying LN pathogenesis. Here we present direct visual evidence from high-power intravital imaging of the local kidney tissue microenvironment in mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx played central roles in LN development. The glomerular homing of T cells was mediated via the direct binding of their CD44 to the hyaluronic acid (HA) component of the endothelial glycocalyx, and glycocalyx-degrading enzymes efficiently disrupted homing. Short-course treatment with either hyaluronidase or heparinase III provided long-term organ protection as evidenced by vastly improved albuminuria and survival rate. This glycocalyx/HA/memory T cell interaction is present in multiple SLE-affected organs and may be therapeutically targeted for SLE complications, including LN.
Hiroyuki Kadoya, Ning Yu, Ina Maria Schiessl, Anne Riquier-Brison, Georgina Gyarmati, Dorinne Desposito, Kengo Kidokoro, Matthew J. Butler, Chaim O. Jacob, János Peti-Peterdi
Depression and anxiety are frequently observed in patients suffering from neuropathic pain. The underlying mechanisms remained unclear. The ventrolateral orbital cortex (VLO) has attracted considerable interest in its role in antidepressive effect in rodents. In the present study, we further investigated the role of the VLO in the anxiodepressive consequences of neuropathic pain in a chronic constriction injury of infraorbital nerve–induced trigeminal neuralgia (TN) mouse model. Elevated plus maze, open field, forced swimming, tail suspension, and sucrose preference tests were used to evaluate anxiodepressive-like behaviors. The results show that chemogenetic activation of bilateral VLO neurons, especially CaMK2A+ pyramidal neurons, blocked the TN-induced anxiodepressive-like behaviors. Chemogenetic and optogenetic activation of VGLUT2+ or inhibition of VGAT+ VLO neurons was sufficient to produce an antianxiodepressive effect in TN mice. Pharmacological activation of D1-like receptors (D1Rs) but not D2Rs in the VLO significantly alleviated TN-induced depressive-like behaviors. Electrophysiological recordings revealed a decreased excitability of VLO excitatory neurons following neuropathic pain. Furthermore, activation of submedius thalamic nucleus–VLO (Sm-VLO) projection mimicked the antianxiodepressive effect of VLO excitation. Conversely, activation of VLO-periaqueductal gray matter (PAG) projection had no effect on TN-induced anxiodepressive behaviors. This study provides a potentially novel mechanism–based therapeutic strategy for the anxiodepressive consequences of neuropathic pain.
Hai-Yan Sheng, Su-Su Lv, Ya-Qi Cai, Wu Shi, Wei Lin, Ting-Ting Liu, Ning Lv, Hong Cao, Ling Zhang, Yu-Qiu Zhang
Seizures can result in a severe hypoperfusion/hypoxic attack that causes postictal memory and behavioral impairments. However, neither postictal changes to microvasculature nor Ca2+ changes in key cell types controlling blood perfusion have been visualized in vivo, leaving essential components of the underlying cellular mechanisms unclear. Here, we use 2-photon microvascular and Ca2+ imaging in awake mice to show that seizures result in a robust vasoconstriction of cortical penetrating arterioles, which temporally mirrors the prolonged postictal hypoxia. The vascular effect was dependent on cyclooxygenase 2, as pretreatment with ibuprofen prevented postictal vasoconstriction. Moreover, seizures caused a rapid elevation in astrocyte endfoot Ca2+ that was confined to the seizure period, and vascular smooth muscle cells displayed a significant increase in Ca2+ both during and following seizures, lasting up to 75 minutes. Our data show enduring postictal vasoconstriction and temporal activities of 2 cell types within the neurovascular unit that are associated with seizure-induced hypoperfusion/hypoxia. These findings support prevention of this event may be a novel and tractable treatment strategy in patients with epilepsy who experience extended postseizure impairments.
Cam Ha T. Tran, Antis G. George, G. Campbell Teskey, Grant R. Gordon
Severe burn injury induces gut barrier dysfunction and subsequently a profound systemic inflammatory response. In the present study, we examined the role of the small intestinal brush border enzyme, intestinal alkaline phosphatase (IAP), in preserving gut barrier function and preventing systemic inflammation after burn wound infection in mice. Mice were subjected to a 30% total body surface area dorsal burn with or without intradermal injection of Pseudomonas aeruginosa. Mice were gavaged with 2000 units of IAP or vehicle at 3 and 12 hours after the insult. We found that both endogenously produced and exogenously supplemented IAP significantly reduced gut barrier damage, decreased bacterial translocation to the systemic organs, attenuated systemic inflammation, and improved survival in this burn wound infection model. IAP attenuated liver inflammation and reduced the proinflammatory characteristics of portal serum. Furthermore, we found that intestinal luminal contents of burn wound–infected mice negatively impacted the intestinal epithelial integrity compared with luminal contents of control mice and that IAP supplementation preserved monolayer integrity. These results indicate that oral IAP therapy may represent an approach to preserving gut barrier function, blocking proinflammatory triggers from entering the portal system, preventing gut-induced systemic inflammation, and improving survival after severe burn injuries.
Fatemeh Adiliaghdam, Paul Cavallaro, Vidisha Mohad, Marianna Almpani, Florian Kühn, Mohammad Hadi Gharedaghi, Mehran Najibi, Laurence G. Rahme, Richard A. Hodin
Increased metabolism distinguishes myofibroblasts or fibrotic lung fibroblasts (fLfs) from the normal lung fibroblasts (nLfs). The mechanism of metabolic activation in fLfs has not been fully elucidated. Furthermore, the antifibrogenic effects of caveolin-1 scaffolding domain peptide CSP/CSP7 involving metabolic reprogramming in fLfs are unclear. We therefore analyzed lactate and succinate levels, as well as the expression of glycolytic enzymes and hypoxia inducible factor-1α (HIF-1α). Lactate and succinate levels, as well as the basal expression of glycolytic enzymes and HIF-1α, were increased in fLfs. These changes were reversed following restoration of p53 or its transcriptional target microRNA-34a (miR-34a) expression in fLfs. Conversely, inhibition of basal p53 or miR-34a increased glucose metabolism, glycolytic enzymes, and HIF-1α in nLfs. Treatment of fLfs or mice having bleomycin- or Ad-TGF-β1–induced lung fibrosis with CSP/CSP7 reduced the expression of glycolytic enzymes and HIF-1α. Furthermore, inhibition of p53 or miR-34a abrogated CSP/CSP7-mediated restoration of glycolytic flux in fLfs in vitro and in mice with pulmonary fibrosis and lacking p53 or miR-34a expression in fibroblasts in vivo. Our data indicate that dysregulation of glucose metabolism in fLfs is causally linked to loss of basal expression of p53 and miR-34a. Treatment with CSP/CSP7 constrains aberrant glucose metabolism through restoration of p53 and miR-34a.
Venkadesaperumal Gopu, Liang Fan, Rashmi S. Shetty, M.R. Nagaraja, Sreerama Shetty
Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase that comprises a scaffold, a catalytic, and multiple regulatory subunits and has been shown to be important in the expression of autoimmunity. We considered that a distinct subunit may account for the decreased production of IL-2 in people and mice with systemic autoimmunity. We show that the regulatory subunit PPP2R2D is increased in T cells from people with systemic lupus erythematosus and regulates IL-2 production. Mice lacking PPP2R2D only in T cells produce more IL-2 because the IL-2 gene and genes coding for IL-2–enhancing transcription factors remain open, while the levels of the enhancer phosphorylated CREB are high. Mice with T cell–specific PPP2R2D deficiency display less systemic autoimmunity when exposed to a TLR7 stimulator. While genes related to Treg function do not change in the absence of PPP2R2D, Tregs exhibit high suppressive function in vitro and in vivo. Because the ubiquitous expression of protein phosphatase 2A cannot permit systemic therapeutic manipulation, the identification of regulatory subunits able to control specific T cell functions opens the way for the development of novel, function-specific drugs.
Wenliang Pan, Amir Sharabi, Andrew Ferretti, Yinfeng Zhang, Catalina Burbano, Nobuya Yoshida, Maria G. Tsokos, George C. Tsokos
Engineering T cells to express chimeric antigen receptors (CARs) specific for antigens on hematological cancers has yielded remarkable clinical responses, but with solid tumors, benefit has been more limited. This may reflect lack of suitable target antigens, immune evasion mechanisms in malignant cells, and/or lack of T cell infiltration into tumors. An alternative approach, to circumvent these problems, is targeting the tumor vasculature rather than the malignant cells directly. CLEC14A is a glycoprotein selectively overexpressed on the vasculature of many solid human cancers and is, therefore, of considerable interest as a target antigen. Here, we generated CARs from 2 CLEC14A-specific antibodies and expressed them in T cells. In vitro studies demonstrated that, when exposed to their target antigen, these engineered T cells proliferate, release IFN-γ, and mediate cytotoxicity. Infusing CAR engineered T cells into healthy mice showed no signs of toxicity, yet these T cells targeted tumor tissue and significantly inhibited tumor growth in 3 mouse models of cancer (Rip-Tag2, mPDAC, and Lewis lung carcinoma). Reduced tumor burden also correlated with significant loss of CLEC14A expression and reduced vascular density within malignant tissues. These data suggest the tumor vasculature can be safely and effectively targeted with CLEC14A-specific CAR T cells, offering a potent and widely applicable therapy for cancer.
Xiaodong Zhuang, Federica Maione, Joseph Robinson, Michael Bentley, Baksho Kaul, Katharine Whitworth, Neeraj Jumbu, Elizabeth Jinks, Jonas Bystrom, Pietro Gabriele, Elisabetta Garibaldi, Elena Delmastro, Zsuzsanna Nagy, David Gilham, Enrico Giraudo, Roy Bicknell, Steven P. Lee
In aging mice, osteoclast number increases in cortical bone but declines in trabecular bone, suggesting that different mechanisms underlie age-associated bone loss in these 2 compartments. Osteocytes produce the osteoclastogenic cytokine RANKL, encoded by Tnfsf11. Tnfsf11 mRNA increases in cortical bone of aged mice, suggesting a mechanism underlying the bone loss. To address this possibility, we aged mice lacking RANKL in osteocytes. Whereas control mice lost cortical bone between 8 and 24 months of age, mice lacking RANKL in osteocytes gained cortical bone during this period. Mice of both genotypes lost trabecular bone with age. Osteoclasts increased with age in cortical bone of control mice but not in RANKL conditional knockout mice. Induction of cellular senescence increased RANKL production in murine and human cell culture models, suggesting an explanation for elevated RANKL levels with age. Overexpression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Finally, elimination of senescent cells from aged mice using senolytic compounds reduced Tnfsf11 mRNA in cortical bone. Our results demonstrate the requirement of osteocyte-derived RANKL for age-associated cortical bone loss and suggest that increased Tnfsf11 expression with age results from accumulation of senescent cells in cortical bone.
Ha-Neui Kim, Jinhu Xiong, Ryan S. MacLeod, Srividhya Iyer, Yuko Fujiwara, Keisha M. Cawley, Li Han, Yonghan He, Jeff D. Thostenson, Elisabeth Ferreira, Robert L. Jilka, Daohong Zhou, Maria Almeida, Charles A. O’Brien
We determined that renal proximal tubular (PT) NF-κB essential modulator (NEMO) plays a direct and critical role in ischemic acute kidney injury (AKI) using mice lacking renal PT NEMO and by targeted renal PT NEMO inhibition with mesoscale nanoparticle–encapsulated NEMO binding peptide (NBP MNP). We subjected renal PT NEMO–deficient mice, WT mice, and C57BL/6 mice to sham surgery or 30 minutes of renal ischemia and reperfusion (IR). C57BL/6 mice received NBP MNP or empty MNP before renal IR injury. Mice treated with NBP MNP and mice deficient in renal PT NEMO were protected against ischemic AKI, having decreased renal tubular necrosis, inflammation, and apoptosis compared with control MNP-treated or WT mice, respectively. Recombinant peptidylarginine deiminase type 4 (rPAD4) targeted kidney PT NEMO to exacerbate ischemic AKI in that exogenous rPAD4 exacerbated renal IR injury in WT mice but not in renal PT NEMO–deficient mice. Furthermore, rPAD4 upregulated proinflammatory cytokine mRNA and NF-κB activation in freshly isolated renal proximal tubules from WT mice but not from PT NEMO–deficient mice. Taken together, our studies suggest that renal PT NEMO plays a critical role in ischemic AKI by promoting renal tubular inflammation, apoptosis, and necrosis.
Sang Jun Han, Ryan M. Williams, Mihwa Kim, Daniel A. Heller, Vivette D’Agati, Marc Schmidt-Supprian, H. Thomas Lee
The spontaneously hypertensive rat (SHR) is a genetic model of primary hypertension with an etiology that includes sympathetic overdrive. To elucidate the neurogenic mechanisms underlying the pathophysiology of this model, we analyzed the dynamic baroreflex response to spontaneous fluctuations in arterial pressure in conscious SHRs, as well as in the Wistar-Kyoto (WKY), the Dahl salt-sensitive, the Dahl salt-resistant, and the Sprague-Dawley rat. Observations revealed the existence of long intermittent periods (lasting up to several minutes) of engagement and disengagement of baroreflex control of heart rate. Analysis of these intermittent periods revealed a predictive relationship between increased mean arterial pressure and progressive baroreflex disengagement that was present in the SHR and WKY strains but absent in others. This relationship yielded the hypothesis that a lower proportion of engagement versus disengagement of the baroreflex in SHR compared with WKY contributes to the hypertension (or increased blood pressure) in SHR compared with WKY. Results of experiments using sinoaortic baroreceptor denervation were consistent with the hypothesis that dysfunction of the baroreflex contributes to the etiology of hypertension in the SHR. Thus, this study provides experimental evidence for the roles of the baroreflex in long-term arterial pressure regulation and in the etiology of primary hypertension in this animal model.
Feng Gu, E. Benjamin Randall, Steven Whitesall, Kimber Converso-Baran, Brian E. Carlson, Gregory D. Fink, Daniel E. Michele, Daniel A. Beard
Preexisting humoral immunity to recombinant adeno-associated virus (AAV) vectors restricts the treatable patient population and efficacy of human gene therapies. Approaches to clear neutralizing antibodies (NAbs), such as plasmapheresis and immunosuppression, are either ineffective or cause undesirable side effects. Here, we describe a clinically relevant strategy to rapidly and transiently degrade NAbs before AAV administration using an IgG-degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaved IgG in dog, monkey, and human antisera. Prophylactically administered IdeZ cleaved circulating human IgG in mice and prevented AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescued AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaved NAbs and rescued AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a potentially new paradigm for expanding the prospective patient cohort and improving efficacy of AAV gene therapy.
Zachary C. Elmore, Daniel K. Oh, Katherine E. Simon, Marco M. Fanous, Aravind Asokan
Hidradenitis suppurativa (HS) is a debilitating chronic inflammatory skin disease characterized by chronic abscess formation and development of multiple draining sinus tracts in the groin, axillae, and perineum. Using proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered on IFN-γ, IL-36, and TNF, with lesser contribution from IL-17A. We further identified B cells and plasma cells, with associated increases in immunoglobulin production and complement activation, as pivotal players in HS pathogenesis, with Bruton’s tyrosine kinase (BTK) and spleen tyrosine kinase (SYK) pathway activation as a central signal transduction network in HS. These data provide preclinical evidence to accelerate the path toward clinical trials targeting BTK and SYK signaling in moderate-to-severe HS.
Johann E. Gudjonsson, Lam C. Tsoi, Feiyang Ma, Allison C. Billi, K.R. van Straalen, A.R.J.V. Vossen, H.H. van der Zee, Paul W. Harms, Rachael Wasikowski, Christine M. Yee, Syed M. Rizvi, Xianying Xing, Enze Xing, Olesya Plazyo, Chang Zeng, Matthew T. Patrick, Margaret M. Lowe, Richard E. Burney, Jeffrey H. Kozlow, Jill R. Cherry-Bukowiec, Yanyun Jiang, Joseph Kirma, Stephan Weidinger, Kelly C. Cushing, Michael D. Rosenblum, Celine Berthier, Amanda S. MacLeod, John J. Voorhees, Fei Wen, J. Michelle Kahlenberg, Emanual Maverakis, Robert L. Modlin, Errol P. Prens
Hidradenitis suppurativa (HS) is a highly prevalent, morbid inflammatory skin disease with limited treatment options. The major cell types and inflammatory pathways in skin of patients with HS are poorly understood, and which patients will respond to TNF-α blockade is currently unknown. We discovered that clinically and histologically healthy appearing skin (i.e., nonlesional skin) is dysfunctional in patients with HS with a relative loss of immune regulatory pathways. HS skin lesions were characterized by quantitative and qualitative dysfunction of type 2 conventional dendritic cells, relatively reduced regulatory T cells, an influx of memory B cells, and a plasma cell/plasmablast infiltrate predominantly in end-stage fibrotic skin. At the molecular level, there was a relative bias toward the IL-1 pathway and type 1 T cell responses when compared with both healthy skin and psoriatic patient skin. Anti–TNF-α therapy markedly attenuated B cell activation with minimal effect on other inflammatory pathways. Finally, we identified an immune activation signature in skin before anti–TNF-α treatment that correlated with subsequent lack of response to this modality. Our results reveal the fundamental immunopathogenesis of HS and provide a molecular foundation for future studies focused on stratifying patients based on likelihood of clinical response to TNF-α blockade.
Margaret M. Lowe, Haley B. Naik, Sean Clancy, Mariela Pauli, Kathleen M. Smith, Yingtao Bi, Robert Dunstan, Johann E. Gudjonsson, Maia Paul, Hobart Harris, Esther Kim, Uk Sok Shin, Richard Ahn, Wilson Liao, Scott L. Hansen, Michael D. Rosenblum
BACKGROUND Hyperglycemia, insulin insensitivity, and low IGF1 levels in extremely preterm infants are associated with an increased risk of retinopathy of prematurity (ROP), but the interactions are incompletely understood.METHODS In 117 extremely preterm infants, serum glucose levels and parenteral glucose intake were recoded daily in the first postnatal week. Serum IGF1 levels were measured weekly. Mice with oxygen-induced retinopathy alone versus oxygen-induced retinopathy plus streptozotocin-induced hyperglycemia/hypoinsulinemia were assessed for glucose, insulin, IGF1, IGFBP1, and IGFBP3 in blood and liver. Recombinant human IGF1 was injected to assess the effect on glucose and retinopathy.RESULTS The highest mean plasma glucose tertile of infants positively correlated with parenteral glucose intake [r(39) = 0.67, P < 0.0001]. IGF1 plasma levels were lower in the high tertile compared with those in low and intermediate tertiles at day 28 (P = 0.038 and P = 0.03). In high versus lower glucose tertiles, ROP was more prevalent (34 of 39 versus 19 of 39) and more severe (ROP stage 3 or higher; 71% versus 32%). In oxygen-induced retinopathy, hyperglycemia/hypoinsulinemia decreased liver IGF1 expression (P < 0.0001); rh-IGF1 treatment improved normal vascular regrowth (P = 0.027) and reduced neovascularization (P < 0.0001).CONCLUSION In extremely preterm infants, high early postnatal plasma glucose levels and signs of insulin insensitivity were associated with lower IGF1 levels and increased ROP severity. In a hyperglycemia retinopathy mouse model, decreased insulin signaling suppressed liver IGF1 production, lowered serum IGF1 levels, and increased neovascularization. IGF1 supplementation improved retinal revascularization and decreased pathological neovascularization. The data support IGF1 as a potential treatment for prevention of ROP.TRIAL REGISTRATION ClinicalTrials.gov NCT02760472 (Donna Mega).FUNDING This study has been supported by the Swedish Medical Research Council (14940, 4732, 20144-01-3, and 21144-01-3), a Swedish government grant (ALFGB2770), Lund medical faculty grants (ALFL, 11615 and 11601), the Skåne Council Foundation for Research and Development, the Linnéa and Josef Carlsson Foundation, the Knut and Alice Wallenberg Foundation, the NIH/National Eye Institute (EY022275, EY017017, EY017017-13S1, and P01 HD18655), European Commission FP7 project 305485 PREVENT-ROP, Deutsche Forschungsgemeinschaft (CA-1940/1-1), and Stiftelsen De Blindas Vänner.
Bertan Cakir, William Hellström, Yohei Tomita, Zhongjie Fu, Raffael Liegl, Anna Winberg, Ingrid Hansen-Pupp, David Ley, Ann Hellström, Chatarina Löfqvist, Lois E.H. Smith
Staphylococcus aureus is prevalent in surgical site infections (SSI) and leads to death in approximately 1% of patients. Phase IIB/III clinical trial results have demonstrated that vaccination against the iron-regulated surface determinant protein B (IsdB) is associated with an increased mortality rate in patients with SSI. Thus, we hypothesized that S. aureus induces nonneutralizing anti-IsdB antibodies, which facilitate bacterial entry into leukocytes to generate “Trojan horse” leukocytes that disseminate the pathogen. Since hemoglobin (Hb) is the primary target of IsdB, and abundant Hb-haptoglobin (Hb-Hp) complexes in bleeding surgical wounds are normally cleared via CD163-mediated endocytosis by macrophages, we investigated this mechanism in vitro and in vivo. Our results demonstrate that active and passive IsdB immunization of mice renders them susceptible to sepsis following SSI. We also found that a multimolecular complex containing S. aureus protein A–anti-IsdB–IsdB–Hb-Hp mediates CD163-dependent bacterial internalization of macrophages in vitro. Moreover, IsdB-immunized CD163–/– mice are resistant to sepsis following S. aureus SSI, as are normal healthy mice given anti-CD163–neutralizing antibodies. These genetic and biologic CD163 deficiencies did not exacerbate local infection. Thus, anti-IsdB antibodies are a risk factor for S. aureus sepsis following SSI, and disruption of the multimolecular complex and/or CD163 blockade may intervene.
Kohei Nishitani, Masahiro Ishikawa, Yugo Morita, Noriaki Yokogawa, Chao Xie, Karen L. de Mesy Bentley, Hiromu Ito, Stephen L. Kates, John L. Daiss, Edward M. Schwarz
Identification of MHC class I–bound peptides by immunopurification of MHC complexes and subsequent analysis by mass spectrometry is crucial for understanding T cell immunology and immunotherapy. Investigation of the steps for the MHC ligand isolation process revealed biases in widely used isolation techniques toward peptides of lower hydrophobicity. As MHC ligand hydrophobicity correlates positively with immunogenicity, identification of more hydrophobic MHC ligands could potentially lead to more effective isolation of immunogenic peptides as targets for immunotherapies. We solved this problem by use of higher concentrations of acetonitrile for the separation of MHC ligands and their respective complexes. This increased overall MHC ligand identifications by 2-fold, increased detection of cancer germline antigen–derived peptides by 50%, and resulted in profound variations in isolation efficacy between different MHC alleles correlating with the hydrophobicity of their anchor residues. Overall, these insights enabled a more complete view of the immunopeptidome and overcame a systematic underrepresentation of these critical MHC ligands of high hydrophobicity.
Martin G. Klatt, Kyeara N. Mack, Yang Bai, Zita E. H. Aretz, Levy I. Nathan, Sung Soo Mun, Tao Dao, David A. Scheinberg
The Ca2+-binding protein calmodulin has emerged as a pivotal player in tuning Na+ channel function, although its impact in vivo remains to be resolved. Here, we identify the role of calmodulin and the NaV1.5 interactome in regulating late Na+ current in cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA). The mutations rendered the channels incapable of binding calmodulin to the C-terminus. The IQ/AA transgenic mice exhibited normal ventricular repolarization without arrhythmias and an absence of increased late Na+ current. In comparison, transgenic mice expressing a lidocaine-resistant (F1759A) human NaV1.5 demonstrated increased late Na+ current and prolonged repolarization in cardiomyocytes, with spontaneous arrhythmias. To determine regulatory factors that prevent late Na+ current for the IQ/AA mutant channel, we considered fibroblast growth factor homologous factors (FHFs), which are within the NaV1.5 proteomic subdomain shown by proximity labeling in transgenic mice expressing NaV1.5 conjugated to ascorbate peroxidase. We found that FGF13 diminished late current of the IQ/AA but not F1759A mutant cardiomyocytes, suggesting that endogenous FHFs may serve to prevent late Na+ current in mouse cardiomyocytes. Leveraging endogenous mechanisms may furnish an alternative avenue for developing novel pharmacology that selectively blunts late Na+ current.
Jeffrey Abrams, Daniel Roybal, Nourdine Chakouri, Alexander N. Katchman, Richard Weinberg, Lin Yang, Bi-xing Chen, Sergey I. Zakharov, Jessica A. Hennessey, Uma Mahesh R. Avula, Johanna Diaz, Chaojian Wang, Elaine Y. Wan, Geoffrey S. Pitt, Manu Ben-Johny, Steven O. Marx
The emergence of SARS-CoV-2 has created an international health crisis, and small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection owing to low-affinity binding to the murine angiotensin-converting enzyme 2 (ACE2) protein. Here, we evaluated the pathogenesis of SARS-CoV-2 in male and female mice expressing the human ACE2 gene under the control of the keratin 18 promoter (K18). In contrast to nontransgenic mice, intranasal exposure of K18-hACE2 animals to 2 different doses of SARS-CoV-2 resulted in acute disease, including weight loss, lung injury, brain infection, and lethality. Vasculitis was the most prominent finding in the lungs of infected mice. Transcriptomic analysis from lungs of infected animals showed increases in transcripts involved in lung injury and inflammatory cytokines. In the low-dose challenge groups, there was a survival advantage in the female mice, with 60% surviving infection, whereas all male mice succumbed to disease. Male mice that succumbed to disease had higher levels of inflammatory transcripts compared with female mice. To our knowledge, this is the first highly lethal murine infection model for SARS-CoV-2 and should be valuable for the study of SARS-CoV-2 pathogenesis and for the assessment of MCMs.
Joseph W. Golden, Curtis R. Cline, Xiankun Zeng, Aura R. Garrison, Brian D. Carey, Eric M. Mucker, Lauren E. White, Joshua D. Shamblin, Rebecca L. Brocato, Jun Liu, April M. Babka, Hypaitia B. Rauch, Jeffrey M. Smith, Bradley S. Hollidge, Collin Fitzpatrick, Catherine V. Badger, Jay W. Hooper
Fibrosis is the final common pathway in the pathophysiology of most forms of chronic kidney disease (CKD). As treatment of renal fibrosis still remains largely supportive, a refined understanding of the cellular and molecular mechanisms of kidney fibrosis and the development of novel compounds are urgently needed. Whether arginases play a role in the development of fibrosis in CKD is unclear. We hypothesized that endothelial arginase-2 (Arg2) promotes the development of kidney fibrosis induced by unilateral ureteral obstruction (UUO). Arg2 expression and arginase activity significantly increased following renal fibrosis. Pharmacologic blockade or genetic deficiency of Arg2 conferred kidney protection following renal fibrosis, as reflected by a reduction in kidney interstitial fibrosis and fibrotic markers. Selective deletion of Arg2 in endothelial cells (Tie2Cre/Arg2fl/fl) reduced the level of fibrosis after UUO. In contrast, selective deletion of Arg2 specifically in proximal tubular cells (Ggt1Cre/Arg2fl/fl) failed to reduce renal fibrosis after UUO. Furthermore, arginase inhibition restored kidney nitric oxide (NO) levels, oxidative stress, and mitochondrial function following UUO. These findings indicate that endothelial Arg2 plays a major role in renal fibrosis via its action on NO and mitochondrial function. Blocking Arg2 activity or expression could be a novel therapeutic approach for prevention of CKD.
Michael D. Wetzel, Kristen Stanley, Wei Wei Wang, Soumya Maity, Muniswamy Madesh, W. Brian Reeves, Alaa S. Awad
Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin-converting enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an ELISA for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of 2 viral-based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus and a spike pseudotyped viral vector–based assay.
Kento T. Abe, Zhijie Li, Reuben Samson, Payman Samavarchi-Tehrani, Emelissa J. Valcourt, Heidi Wood, Patrick Budylowski, Alan P. Dupuis II, Roxie C. Girardin, Bhavisha Rathod, Jenny H. Wang, Miriam Barrios-Rodiles, Karen Colwill, Allison J. McGeer, Samira Mubareka, Jennifer L. Gommerman, Yves Durocher, Mario Ostrowski, Kathleen A. McDonough, Michael A. Drebot, Steven J. Drews, James M. Rini, Anne-Claude Gingras
BACKGROUND. Left atrial (LA) and left ventricular (LV) remodeling are associated with atrial fibrillation (AF). The prospective associations of impairment in cardiac mechanical function, as assessed by speckle-tracking echocardiography, with incident AF are less clear. METHODS. In the Cardiovascular Health Study, a community-based cohort of older adults, participants free of AF with echocardiograms of adequate quality for speckle tracking were included. We evaluated the associations of indices of cardiac mechanics (LA reservoir strain, LV longitudinal strain, and LV early diastolic strain rate) with incident AF. RESULTS. Of 4341 participants with strain imaging, participants with lower LA reservoir strain were older, had more cardiometabolic risk factors, and had lower renal function at baseline. Over a median follow-up of 10 years, 497 (11.4%) participants developed AF. Compared with the highest quartile of LA reservoir strain, the lowest quartile of LA reservoir strain was associated with higher risk of AF after covariate adjustment, including LA volume and LV longitudinal strain (Hazard Ratio [HR], 1.80; 95% CI, 1.31–2.45; P < 0.001). The association of LA reservoir strain and AF was stronger in subgroups with higher blood pressure, NT-proBNP, and LA volumes. There were no associations of LV longitudinal strain and LV early diastolic strain rate with incident AF after adjustment for LA reservoir strain. CONCLUSION. Lower LA reservoir strain was associated with incident AF, independent of LV mechanics, and with stronger associations in high-risk subgroups. These findings suggest that LA mechanical dysfunction precedes the development of AF. Therapies targeting LA mechanical dysfunction may prevent progression to AF. FUNDING. This research was supported by contracts HHSN268201200036C, HHSN268200800007C, HHSN268201800001C, N01HC55222, N01HC85079, N01HC85080, N01HC85081, N01HC85082, N01HC85083, and N01HC85086 and grants KL2TR001424, R01HL107577, U01HL080295, and U01HL130114 from the NIH’s National Center for Advancing Translational Sciences, and National Heart, Lung, and Blood Institute (NHLBI), with additional contribution from the National Institute of Neurological Disorders and Stroke (NINDS). Additional support was provided by R01AG023629 from the National Institute on Aging (NIA). A full list of principal CHS investigators and institutions can be found at CHS-NHLBI.org.
Ravi B. Patel, Joseph A. Delaney, Mo Hu, Harnish Patel, Jeanette Cheng, John Gottdiener, Jorge R. Kizer, Gregory M. Marcus, Mintu P. Turakhia, Rajat Deo, Susan R. Heckbert, Bruce M. Psaty, Sanjiv J. Shah
Huntington’s disease (HD) is a progressive, autosomal dominant neurodegenerative disorder affecting striatal neurons beginning in young adults with loss of muscle coordination and cognitive decline. Less appreciated is the fact that patients with HD also exhibit cardiac and respiratory dysfunction, including pulmonary insufficiency and cardiac arrhythmias. The underlying mechanism for these symptoms is poorly understood. In the present study we provide insight into the cause of cardiorespiratory dysfunction in HD and identify a potentially novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via posttranslationally modified ryanodine receptor/intracellular calcium release (RyR) channels plays an important role in HD pathology. RyR channels were oxidized, PKA phosphorylated, and leaky in brain, heart, and diaphragm both in patients with HD and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum Ca2+ leak exhibited cognitive dysfunction, decreased parasympathetic tone associated with cardiac arrhythmias, and reduced diaphragmatic contractile function resulting in impaired respiratory function. Defects in cognitive, motor, and respiratory functions were ameliorated by treatment with a novel Rycal small-molecule drug (S107) that fixes leaky RyR. Thus, leaky RyRs likely play a role in neuronal, cardiac, and diaphragmatic pathophysiology in HD, and RyRs are a potential novel therapeutic target.
Haikel Dridi, Xiaoping Liu, Qi Yuan, Steve Reiken, Mohamad Yehya, Leah Sittenfeld, Panagiota Apostolou, Julie Buron, Pierre Sicard, Stefan Matecki, Jérome Thireau, Clement Menuet, Alain Lacampagne, Andrew R. Marks
Posttranslational glutamylation/deglutamylation balance in tubulins influences dendritic maturation and neuronal survival of cerebellar Purkinje neurons (PNs). PNs and some additional neuronal types degenerate in several spontaneous, independently occurring Purkinje cell degeneration (pcd) mice featuring mutant neuronal nuclear protein induced by axotomy (Nna1), a deglutamylase gene. This defective deglutamylase allows glutamylases to form hyperglutamylated tubulins. In pcd, all PNs die during postnatal “adolescence.” Neurons in some additional brain regions also die, mostly later than PNs. We show in laser capture microdissected single PNs, in cerebellar granule cell neuronal clusters, and in dissected hippocampus and substantia nigra that deglutamase mRNA and protein were virtually absent before pcd PNs degenerated, whereas glutaminase mRNA and protein remained normal. Hyperglutamylated microtubules and dimeric tubulins accumulated in pcd PNs and were involved in pcd PN death by glutamylase/deglutamylase imbalance. Importantly, treatment with a microtubule depolymerizer corrected the glutamylation/deglutamylation ratio, increasing PN survival. Further, before onset of neuronal death, pcd PNs displayed prominent basal polylisosomal masses rich in ER. We propose a “seesaw” metamorphic model summarizing mutant Nna1-induced tubulin hyperglutamylation, the pcd’s PN phenotype, and report that the neuronal disorder involved ER stress, unfolded protein response, and protein synthesis inhibition preceding PN death by apoptosis/necroptosis.
Jianxue Li, Evan Y. Snyder, Fenny H.F. Tang, Renata Pasqualini, Wadih Arap, Richard L. Sidman
There is no cure for the more than 270 million people chronically infected with HBV. Nucleos(t)ide analogs (NUCs), the mainstay of anti-HBV treatment, block HBV reverse transcription. NUCs do not eliminate the intranuclear covalently closed circular DNA (cccDNA), from which viral RNAs, including pregenomic RNA (pgRNA), are transcribed. A key gap in designing a cure is understanding how NUCs affect HBV replication and transcription because serum markers yield an incomplete view of intrahepatic HBV. We applied single-cell laser capture microdissection and droplet digital PCR to paired liver biopsies collected from 5 HBV/HIV-coinfected persons who took NUCs over 2–4 years. From biopsy 1 to 2, proportions of HBV-infected hepatocytes declined with adherence to NUC treatment (P < 0.05); we extrapolated that eradication of HBV will take over 10 decades with NUCs in these participants. In individual hepatocytes, pgRNA levels diminished 28- to 73-fold during NUC treatment, corresponding with decreased tissue HBV core antigen staining (P < 0.01). In 4 out of 5 participants, hepatocytes with cccDNA but undetectable pgRNA (transcriptionally inactive) were present, and these were enriched in 3 participants during NUC treatment. Further work to unravel mechanisms of cccDNA transcriptional inactivation may lead to therapies that can achieve this in all hepatocytes, resulting in a functional cure.
Ashwin Balagopal, Tanner Grudda, Ruy M. Ribeiro, Yasmeen S. Saad, Hyon S. Hwang, Jeffrey Quinn, Michael Murphy, Kathleen Ward, Richard K. Sterling, Yang Zhang, Alan S. Perelson, Mark S. Sulkowski, William O. Osburn, Chloe L. Thio