We determined that renal proximal tubular (PT) NFκB essential modulator (NEMO) plays a direct and critical role in ischemic acute kidney injury (AKI) utilizing using mice lacking renal PT NEMO and by targeted renal PT NEMO inhibition with mesoscale nanoparticle encapsulated NEMO binding peptide (MNP NBP). We subjected renal PT NEMO deficient mice, wild type (WT) mice and C57BL/6 mice to sham surgery or 30 min renal ischemia and reperfusion (IR). C57BL/6 mice received NBP MNP or empty MNP before renal IR injury. Mice treated with MNP NBP and mice deficient in renal PT NEMO were protected against ischemic AKI with decreased renal tubular necrosis, inflammation and apoptosis compared to control MNP treated or WT mice, respectively. Recombinant peptidylarginine deiminase type-4 (rPAD4) targets kidney PT NEMO to exacerbate ischemic AKI as exogenous rPAD4 exacerbated renal IR injury in WT mice but not in renal proximal tubule NEMO deficient mice. Furthermore, rPAD4 upregulated proinflammatory cytokine mRNA and NFκB 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 as well as necrosis.
Sang Jun Han, Ryan M. williams, Mihwa Kim, Daniel A. Heller, Vivette D'Agati, Marc Schmidt-Supprian, H. Thomas Lee
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 development of fibrosis in CKD is unclear. We hypothesize 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. Pharmacological 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/Arg2flox/flox) reduced the level of fibrosis after UUO. In contrast, selective deletion of Arg2 specifically in proximal tubular cells (Ggt1Cre/Arg2flox/flox) 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 Wetzel, Kristen Stanley, Wei Wei Wang, Soumya Maity, Muniswamy Madesh, W. Brian Reeves, Alaa S. Awad
Pre-existing humoral immunity to recombinant adeno-associated viral (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 prior to AAV administration using an IgG degrading enzyme (IdeZ). We demonstrate that recombinant IdeZ efficiently cleaves IgG in dog, monkey and human antisera. Prophylactically administered IdeZ cleaves circulating, human IgG in mice and prevents AAV neutralization in vivo. In macaques, a single intravenous dose of IdeZ rescues AAV transduction by transiently reversing seropositivity. Importantly, IdeZ efficiently cleaves NAbs and rescues AAV transduction in mice passively immunized with individual human donor sera representing a diverse population. Our antibody clearance approach presents a 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
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 4,341 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 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 (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 mechanical dysfunction of the LA precedes the development of AF. Therapies targeting LA mechanical dysfunction may prevent progression to AF.
Ravi B. Patel, Joseph A. Delaney, Mo Hu, Harnish Patel, Jeanette Y. 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 HD patients 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 novel therapeutic target. We now show that intracellular calcium (Ca2+) leak via post-translationally 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 in both HD patients and in a murine model of HD (Q175). HD mice (Q175) with endoplasmic reticulum (ER) 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 identify RyRs as a potential novel therapeutic target.
Haikel Dridi, Xiaoping Liu, Qi Yuan, Steve Reiken, Yehya Mohamad, Leah R. Sittenfeld, Panagiota Apostolou, Julie Buron, Pierre Sicard, Stefan Matecki, Jérôme Thireau, Clement Menuet, Alain Lacampagne, Andrew R. Marks
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 towards 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 (ACN) for the separation of MHC ligands and their respective complexes. This increased overall MHC ligand identifications by 2-fold, 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 on 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
Protein phosphatase 2A is a ubiquitously expressed serine/threonine phosphatase which 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 interleukin-2 (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 and 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 regulatory T cell function do not change in the absence of PPP2R2D, regulatory T cells 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 P. Ferretti, Yinfeng Zhang, Catalina Burbano, Nobuya Yoshida, Maria G. Tsokos, George C. Tsokos
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 enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of two 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 Wang, Miriam Barrios-Rodiles, Karen Colwill, Allison 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
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 IQ/AA mutant, 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 find FGF13 diminishes 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 M. Abrams, Daniel Roybal, Nourdine Chakouri, Alex N. Katchman, Richard L. 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
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 two 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 24-month-old control mice had lower cortical bone compared to 8-month-old controls, mice lacking RANKL in osteocytes gained cortical bone from 8 to 24 months of age. 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. Over-expression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Lastly, 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
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 new mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx play 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 it. 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
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. Utilizing proteomic and transcriptomic approaches, we characterized the inflammatory responses in HS in depth, revealing immune responses centered around 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 BTK and SYK pathway activation as a central signal transduction networks in HS. These data provide preclinical evidence to accelerate the path towards clinical trials targeting BTK and SYK signaling in moderate to severe HS.
Johann E. Gudjonsson, Lam C. Tsoi, Feiyang Ma, Allison C. Billi, Kelsey R. van Straalen, Allard R.J.V. Vossen, H.H. Zee, Paul W. Harms, Rachael Wasikowski, Christine M. Yee, Syed Monem 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 C. Berthier, Amanda S. MacLeod, John J. Voorhees, Fei Wen, J. Michelle Kahlenberg, Emanual Maverakis, Robert L. Modlin, Errol P. Prens
The emergence of SARS-CoV-2 has created an international health crisis. Small animal models mirroring SARS-CoV-2 human disease are essential for medical countermeasure (MCM) development. Mice are refractory to SARS-CoV-2 infection due 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 promotor. In contrast to non-transgenic mice, intranasal exposure of K18-hACE2 animals to two 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 revealed increases in transcripts involved in lung injury and inflammatory cytokines. In the lower 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 to female mice. This is the first highly lethal murine infection model for SARS-CoV-2. The K18-hACE2 murine model will be valuable for the study of SARS-CoV-2 pathogenesis and 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
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. Further, the anti-fibrogenic effects of caveolin-1 scaffolding domain peptide CSP/CSP7 involve metabolic reprogramming in fLfs is unclear. We therefore analyzed lactate and succinate levels, and the expression of glycolytic enzymes, and hypoxia inducible factor-1alpha (HIF-1α). Lactate and succinate levels as well as the basal expression of glycolytic enzymes and HIF-1α αwere 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 TGF-beta1-induced lung fibrosis with CSP/CSP7, reduced the expression of glycolytic enzymes and HIF-1α. Further, 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 Shetty, MR Nagaraja, Sreerama Shetty
Hidradenitis suppurativa (HS) is a highly prevalent and morbid inflammatory skin disease with limited treatment options. The major cell types and inflammatory pathways in skin of HS patients are poorly understood. In addition, it is currently unknown which patients will respond to TNFα blockade. Herein, we comprehensively elucidate and functionally define the immune cell infiltrate and major inflammatory pathways in HS skin, before and after anti-TNFα therapy. We discovered that clinically and histologically healthy appearing skin (i.e., nonlesional skin) is dysfunctional in HS patients with a relative loss of immune regulatory pathways. At the cellular level, HS skin lesions were characterized by quantitative and qualitative dysfunction of type 2 dendritic cells (cDC2s), relatively reduced regulatory T cells (Tregs), 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 towards the IL-1 pathway and type 1 T cell responses when compared to both healthy skin and skin from psoriasis patients. Anti-TNFα therapy significantly attenuated B cell activation with minimal effect on other inflammatory pathways. Finally, we identified an immune activation signature in skin prior to anti-TNFα treatment that correlated with subsequent lack of response to this modality. Taken together, 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 TNFα blockade.
Margaret M. Lowe, Haley B. Naik, Sean Clancy, Mariela Pauli, Kathleen M. Smith, Yingtao Bi, Robert Dunstan, Johann Gudjonsson, Maia Paul, Hobart W. Harris, Esther A. Kim, Uk Sok Shin, Richard Ahn, Wilson Liao, Scott L. Hansen, Michael Rosenblum