HIV is a major driver of Tuberculosis (TB) reactivation. Depletion of CD4+ T cells is assumed to be the basis behind TB reactivation in individuals with latent tuberculosis Infection (LTBI) co-infected with human immunodeficiency virus (HIV). Non-human primates (NHPs) coinfected with a mutant simian immunodeficiency virus (SIVΔGY), that does not cause depletion of tissue CD4+ T cells during infection, failed to reactivate TB. To investigate the contribution of CD4+ T cell depletion relative to other mechanisms of SIV-induced reactivation of LTBI, we used CD4R1 antibody to deplete CD4+ T cells in animals with LTBI without lentiviral infection. We showed that the mere depletion of CD4+ T cells during LTBI was insufficient in generating reactivation of LTBI. Instead, direct cytopathic effects of SIV resulting in chronic immune activation, along with the altered effector T cell phenotypes and dysregulated T cell homeostasis, were likely mediators of reactivation of LTBI. These results revealed important implications for controlling TB in the HIV co-infected individuals.
Allison N. Bucşan, Ayan Chatterjee, Dhiraj K. Singh, Taylor W. Foreman, Tae-Hyung Lee, Breanna Threeton, Melanie G. Kirkpatrick, Mushtaq Ahmed, Nadia Golden, Xavier Alvarez, James A. Hoxie, Smriti Mehra, Jyothi Rengarajan, Shabaana A. Khader, Deepak Kaushal
During developmental angiogenesis blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether and how cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death-signaling pathway, regulates both cell death via apoptosis and necroptosis. Here we show that expression of Casp-8 in endothelial cells (ECs) was required for proper postnatal retina angiogenesis. EC specific Casp-8 knockout pups (Casp-8ECko) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting and migration independent of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 mitogen-activated protein kinase (MAPK) downstream of receptor-interacting serine/threonine- protein kinase 3 (RIPK3) and destabilization of VE-cadherin at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR), resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-8ECko pups. Taken together, we describe that Casp-8 acts in a cell-death independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP.
Nathalie Tisch, Aida Freire-Valls, Rosario Yerbes, Isidora Paredes, Silvia La Porta, Xiaohong Wang, Rosa Martín-Pérez, Laura Castro, Wendy Wei-Lynn Wong, Leigh Coultas, Boris Strilic, Hermann-Josef Gröne, Thomas Hielscher, Carolin Mogler, Ralf Adams, Peter Heiduschka, Lena Claesson-Welsh, Massimiliano Mazzone, Abelardo López-Rivas, Thomas Schmidt, Hellmut G. Augustin, Carmen Ruiz de Almodovar
Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH) defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO-signaling. We identified two pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin-toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.
Michael Hugelshofer, Raphael M. Buzzi, Christian A. Schaer, Henning Richter, Kevin Akeret, Vania Anagnostakou, Leila Mahmoudi, Raphael Vaccani, Florence Vallelian, Jeremy W. Deuel, Peter W. Kronen, Zsolt Kulcsar, Luca Regli, Jin Hyen Baek, Ivan S. Pires, Andre F. Palmer, Matthias Dennler, Rok Humar, Paul W. Buehler, Patrick R. Kircher, Emanuela Keller, Dominik J. Schaer
Overexpression of myo-inositol oxygenase (MIOX), a proximal tubular enzyme, exacerbates cellular redox injury in acute kidney injury (AKI). Ferroptosis, a newly coined term associated with lipid hydroperoxidation, plays a critical role in the pathogenesis of AKI. Whether or not MIOX exacerbates tubular damage by accelerating ferroptosis in Cisplatin-induced AKI remains elusive. Cisplatin-treated HK-2 cells exhibited notable cell death, which was reduced by ferroptosis inhibitors. Also, alterations in various ferroptosis metabolic sensors, including lipid hydroperoxidation, glutathione peroxidase 4 (GPX4) activity, NADPH and reduced glutathione (GSH) levels, and ferritinophagy, were observed. These perturbations were accentuated by MIOX overexpression, while ameliorated by MIOX knockdown. Likewise, Cisplatin-treated CD1 mice exhibited tubular damage and derangement of renal physiological parameters, which was alleviated by Ferrostatin-1 (Fer-1), a ferroptosis inhibitor. To investigate the relevance of MIOX to ferroptosis, Wild-type (WT) mice, MIOX-overexpressing transgenic (MIOX-TG) mice and MIOX knockout (MIOX-KO) mice were subjected to Cisplatin treatment. In comparison to Cisplatin-treated WT mice, Cisplatin-treated MIOX-TG mice had more severe renal pathological changes and perturbations in ferroptosis metabolic sensors, which were minimal in Cisplatin-treated MIOX-KO mice. In conclusion, these findings indicate that ferroptosis, an integral process in the pathogenesis of Cisplatin-induced AKI, is modulated by the expression profile of MIOX.
Fei Deng, Isha Sharma, Yingbo Dai, Ming Yang, Yashpal S. Kanwar
The interleukin-3 receptor alpha subunit, CD123, is expressed on many hematologic malignancies including acute myeloid leukemia (AML) and blastic plasmacytoid dendritic cell neoplasm (BPDCN). Tagraxofusp (SL-401) is a CD123-targeted therapy consisting of interleukin-3 fused to a truncated diphtheria toxin payload. Factors influencing response to tagraxofusp other than CD123 expression are largely unknown. We interrogated tagraxofusp resistance in patients and experimental models and found that it was not associated with CD123 loss. Rather, resistant AML and BPDCN cells frequently acquired deficiencies in the diphthamide synthesis pathway, impairing tagraxofusp’s ability to ADP-ribosylate cellular targets. Expression of DPH1, encoding a diphthamide pathway enzyme, was reduced by DNA CpG methylation in resistant cells. Treatment with the DNA methyltransferase inhibitor azacitidine restored DPH1 expression and tagraxofusp sensitivity. We also developed a drug-dependent ADP-ribosylation assay in primary cells that correlated with tagraxofusp activity and may represent an additional novel biomarker. As predicted by these results and our observation that resistance also increased mitochondrial apoptotic priming, we found that the combination of tagraxofusp and azacitidine was effective in patient-derived xenografts treated in vivo. These data have important implications for clinical use of tagraxofusp and led to a phase 1 study combining tagraxofusp and azacitidine in myeloid malignancies.
Katsuhiro Togami, Timothy Pastika, Jason Stephansky, Mahmoud Ghandi, Amanda L. Christie, Kristen L. Jones, Carl A. Johnson, Ross W. Lindsay, Christopher L. Brooks, Anthony Letai, Jeffrey W. Craig, Olga Pozdnyakova, David M. Weinstock, Joan Montero, Jon C. Aster, Cory M. Johannessen, Andrew A. Lane
Asthma is a heterogeneous syndrome that has been subdivided into physiological phenotypes and molecular endotypes. The most specific phenotypic manifestation of asthma is indirect airway hyperresponsiveness (AHR), and a prominent molecular endotype is the presence of type-2 inflammation. The underlying basis for type-2 inflammation and its relationship to AHR are incompletely understood. We assessed the expression of type-2 cytokines in the airways of subjects with and without asthma who were extensively characterized for AHR. Using quantitative morphometry of the airway wall, we identified a shift in mast cells from the submucosa to the airway epithelium specifically associated with both type-2 inflammation and indirect AHR. Using ex vivo modeling of primary airway epithelial cells in organotypic co-culture with mast cells, we have shown that epithelial-derived IL-33 uniquely induced type-2 cytokines in mast cells, which regulated the expression of epithelial IL33 in a feedforward loop. This feedforward loop was accentuated in epithelial cells derived from subjects with asthma. These results demonstrate that type-2 inflammation and indirect AHR in asthma are related to a shift in mast cell infiltration to the airway epithelium, and that mast cells cooperate with epithelial cells through IL-33 signaling to regulate type-2 inflammation.
Matthew C. Altman, Ying Lai, James D. Nolin, Sydney Long, Chien-Chang Chen, Adrian M. Piliponsky, William A. Altemeier, Megan Larmore, Charles W. Frevert, Michael S. Mulligan, Steven F. Ziegler, Jason S. Debley, Michael C. Peters, Teal S. Hallstrand
Patients with paroxysmal nocturnal hemoglobinuria (PNH) have a clonal population of blood cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins, resulting from a mutation in the X-linked gene PIGA. Here we report on a set of patients in whom PNH results instead from biallelic mutation of PIGT on chromosome 20. These PIGT-PNH patients have clinically typical PNH, but they have in addition prominent auto-inflammatory features, including recurrent attacks of aseptic meningitis. In all these patients we find a germ-line point mutation in one PIGT allele, whereas the other PIGT allele is removed by somatic deletion of a 20q region comprising maternally imprinted genes implicated in myeloproliferative syndromes. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. From studies of patients’ leukocytes and of PIGT-knockout THP-1 cells we show that, through increased IL-1β secretion, activation of the lectin pathway of complement and generation of C5b-9 complexes, free GPI is the agent of auto-inflammation. Eculizumab treatment abrogates not only intravascular hemolysis, but also auto-inflammation. Thus, PIGT-PNH differs from PIGA-PNH both in the mechanism of clonal expansion and in clinical manifestations.
Britta Höchsmann, Yoshiko Murakami, Makiko Osato, Alexej Knaus, Michi Kawamoto, Norimitsu Inoue, Tetsuya Hirata, Shogo Murata, Markus Anliker, Thomas Eggermann, Marten Jäger, Ricarda Floettmann, Alexander Höellein, Sho Murase, Yasutaka Ueda, Jun-ichi Nishimura, Yuzuru Kanakura, Nobuo Kohara, Hubert Schrezenmeier, Peter M. Krawitz, Taroh Kinoshita
The parathyroid hormone receptor (PTH1R) mediates the biologic actions of parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP). Here, we showed that salt inducible kinases (SIKs) are key kinases that control the skeletal actions downstream of PTH1R and that this GPCR, when activated, inhibited cellular SIK activity. Sik gene deletion led to phenotypic changes that were remarkably similar to models of increased PTH1R signaling. In growth plate chondrocytes, PTHrP inhibited SIK3 and ablation of this kinase in proliferating chondrocytes rescued perinatal lethality of PTHrP-null mice. Combined deletion of Sik2/Sik3 in osteoblasts and osteocytes led to a dramatic increase in bone mass that closely resembled the skeletal and molecular phenotypes observed when these bone cells express a constitutively active PTH1R that causes Jansen’s metaphyseal chondrodysplasia. Finally, genetic evidence demonstrated that class IIa HDACs were key PTH1R-regulated SIK substrates in both chondrocytes and osteocytes. Taken together, our findings established that SIK inhibition is central to PTH1R action in bone development and remodeling. Furthermore, this work highlighted the key role of cAMP-regulated salt inducible kinases downstream of GPCR action.
Shigeki Nishimori, Maureen J. O'Meara, Christian Castro, Hiroshi Noda, Murat Cetinbas, Janaina da Silva Martins, Ugur Ayturk, Daniel J. Brooks, Michael Bruce, Mizuki Nagata, Wanida Ono, Christopher J. Janton, Mary L. Bouxsein, Marc Foretz, Rebecca Berdeaux, Ruslan I. Sadreyev, Thomas J. Gardella, Harald Jüppner, Henry M. Kronenberg, Marc N. Wein
The Toll-Like Receptor 8 (TLR8) has an important role in innate immune responses to RNA viral infections including respiratory syncytial virus (RSV). We reported previously that TLR8 expression was increased directly by the tumor suppressor and transcription factor p53 via a single nucleotide polymorphism (SNP: rs3761624) in the TLR8 promoter, thereby placing TLR8 in the p53/immune axis. Because this SNP is in linkage disequilibrium with other SNPs associated with several infectious diseases, we addressed the combined influence of p53 and the SNP on downstream inflammatory signaling in response to a TLR8 cognate ssRNA ligand. Using human primary lymphocytes, p53 induction by chemotherapeutic agents such as ionizing radiation caused SNP-dependent synergistic increases in IL-6 following incubation with an ssRNA ligand, as well as TLR8 RNA and protein expression along with p53 binding at the TLR-p53 SNP site. Because TLR8 is X-linked, the increases were generally reduced in heterozygous females. We found a corresponding association of the p53-responsive allele with RSV disease severity in infants hospitalized with RSV infection. We conclude that p53 can strongly influence TLR8 mediated immune responses and that knowledge of the p53 responsive SNP can inform diagnosis and prognosis of RSV disease and other diseases that might have a TLR8 component, including cancer.
Daniel Menendez, Joyce Snipe, Jacqui Marzec, Cynthia L. Innes, Fernando P. Polack, Mauricio Caballero, Shepherd H. Schurman, Steven R. Kleeberger, Michael A. Resnick
Molecular heterogeneity of endothelial cells underlies their highly-specialized functions during changing physiological conditions within diverse vascular beds. For example, placental spiral arteries (SAs) undergo remarkable remodeling to meet the ever-growing demands of the fetus—a process which is deficient in preeclampsia. The extent to which maternal endothelial cells coordinate with immune cells and pregnancy hormones to promote SA remodeling remains largely unknown. Here we found that remodeled SAs expressed the lymphatic markers PROX1, LYVE1, and VEGFR3, mimicking lymphatic identity. Uterine natural killer (uNK) cells, which are required for SA remodeling and secrete VEGFC, were both sufficient and necessary for VEGFR3 activation in vitro and in mice lacking uNK cells, respectively. Using Flt4Chy/+ mice with kinase inactive VEGFR3 and Vegfcfl/fl;Vav1-Cre mice, we demonstrated that SA remodeling required VEGFR3 signaling, and that disrupted maternal VEGFR3 signaling contributed to late-gestation fetal growth restriction. Collectively, we identified a novel instance of lymphatic mimicry by which maternal endothelial cells promote SA remodeling, furthering our understanding of the vascular heterogeneity employed for the mitigation of pregnancy complications such as fetal growth restriction and preeclampsia.
John B. Pawlak, László Bálint, Lillian Lim, Wanshu Ma, Reema B. Davis, Zoltan Benyo, Michael J. Soares, Guillermo Oliver, Mark L. Kahn, Zoltán Jakus, Kathleen M. Caron
BACKGROUND. Impaired T-cell immunity in transplant recipients is associated with infection-related morbidity and mortality. We recently reported the successful use of adoptive T-cell therapy (ACT) against drug-resistant/recurrent cytomegalovirus in solid-organ transplant recipients. METHODS. In the present study, we employed high-throughput T-cell receptor Vβ sequencing and T-cell functional profiling to delineate the impact of ACT on T-cell repertoire remodelling in the context of pre-therapy immunity and ACT products. RESULTS. These analyses indicated that a clinical response was coincident with significant changes in the T-cell receptor Vβ landscape post-therapy. This restructuring was associated with the emergence of effector memory (EM) T cells in responding patients, while non-responders displayed dramatic pre-therapy T-cell expansions with minimal change following ACT. Furthermore, immune reconstitution included both adoptively transferred clonotypes and endogenous clonotypes not detected in the ACT products. CONCLUSION. These observations demonstrate that immune control following ACT requires significant repertoire remodelling, which may be impaired in non-responders due to the pre-existing immune environment. Immunological interventions that can modulate this environment may improve clinical outcomes.
Corey Smith, Dillon Corvino, Leone Beagley, Sweera Rehan, Michelle A. Neller, Pauline Crooks, Katherine K. Matthews, Matthew Solomon, Laetitia Le Texier, Scott Campbell, Ross S. Francis, Daniel Chambers, Rajiv Khanna
We hypothesized that the store operated calcium entry (SOCE) channel, Orai1, participates in the activation of T-helper (Th17) cells and influences renal injury. In rats following renal ischemia/reperfusion (I/R), there was a rapid and sustained influx of Orai1+ CD4 T-cells and IL17 expression was restricted to Orai1-positive cells. When kidney CD4+ cells of post-AKI rats were stimulated with angiotensin II and elevated Na+ (10-7M/170 mM) in vitro, there was an enhanced response in intracellular Ca2+ and IL17 expression, which was blocked by SOCE inhibitors 2APB, YM58483/BTP2, or AnCoA4. In vivo, YM58343/BTP2 (1 mg ∙ kg-1) attenuated IL17+ cell activation, inflammation and severity of AKI following either I/R or intramuscular glycerol injection. Rats treated with high-salt diet (5-9 weeks post I/R) manifested progressive disease indicated by enhanced inflammation, fibrosis and impaired renal function. These responses were significantly attenuated by YM58343/BTP2. In peripheral blood of critically ill patients, Orai1+ cells were significantly elevated by ~10-fold and Th17 cells were elevated by ~4 fold in AKI vs non-AKI patients. Further, in vitro stimulation of CD4+ cells from AKI patients increased IL17, which was blocked by SOCE inhibitors. These data suggest that Orai1 SOCE is a potential therapeutic target in AKI and CKD progression.
Purvi Mehrotra, Michael Sturek, Javier A. Neyra, David P. Basile
Background: Idiopathic multicentric Castleman disease (iMCD) is a hematologic illness involving cytokine-induced lymphoproliferation, systemic inflammation, cytopenias, and life-threatening multi-organ dysfunction. The molecular underpinnings of interleukin-6(IL-6)-blockade refractory patients remain unknown; no targeted therapies exist. In this study, we searched for therapeutic targets in IL-6-blockade refractory iMCD patients with the thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, organomegaly (TAFRO) clinical subtype. Methods: We analyzed tissues and blood samples from three IL-6-blockade refractory iMCD-TAFRO patients. Cytokine panels, quantitative serum proteomics, flow cytometry of PBMCs, and pathway analyses were employed to identify novel therapeutic targets. To confirm elevated mTOR signaling, a candidate therapeutic target from the above assays, immunohistochemistry was performed for phosphorylated S6, a read-out of mTOR activation, in three iMCD lymph node tissue samples and controls. Proteomic, immunophenotypic, and clinical response assessments were performed to quantify the effects of administration of the mTOR inhibitor, sirolimus. Results: Studies of three IL-6-blockade refractory iMCD cases revealed increased CD8+ T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus significantly attenuated CD8+ T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all three patients with durable and ongoing remissions of 66, 19, and 19 months. Conclusion: This precision medicine approach identifies PI3K/Akt/mTOR signaling as the first pharmacologically-targetable pathogenic process in IL-6-blockade refractory iMCD. Prospective evaluation of sirolimus in treatment-refractory iMCD is planned (NCT03933904). Funding: Castleman’s Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and National Heart Lung and Blood Institute.
David C. Fajgenbaum, Ruth-Anne Langan, Alberto Sada Japp, Helen L. Partridge, Sheila K. Pierson, Amrit Singh, Daniel J. Arenas, Jason R. Ruth, Christopher S. Nabel, Katie Stone, Mariko Okumura, Anthony Schwarer, Fábio Freire Jose, Nelson Hamerschlak, Gerald B. Wertheim, Michael B. Jordan, Adam D. Cohen, Vera Krymskaya, Arthur Rubenstein, Michael R. Betts, Taku Kambayashi, Frits van Rhee, Thomas S. Uldrick
Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site Ser651. Active PDE then hydrolyzes anti-inflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site in PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in athero-prone regions of arteries, and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also unexpectedly stabilized the PP2A-B55α complex. The integrin-regulated, pro-atherosclerotic transcription factor Yap is also dephosphorylated and activated through this pathway. PDE4D5 therefore mediates matrix-specific regulation of EC phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex with other targets. These results are likely to have widespread consequences for control of cell function by integrins.
Sanguk Yun, Rui Hu, Melanie E. Schwaemmle, Alexander N. Scherer, Zhenwu Zhuang, Anthony J. Koleske, David C. Pallas, Martin A. Schwartz
Deciphering novel pathways regulating liver lipid content has profound implications for understanding the pathophysiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Recent evidence suggests that the nuclear envelope is a site of regulation of lipid metabolism but there is limited appreciation of the responsible mechanisms and molecular components within this organelle. We showed that conditional hepatocyte deletion of the inner nuclear membrane protein lamina-associated polypeptide 1 (LAP1) caused defective VLDL secretion and steatosis, including intranuclear lipid accumulation. LAP1 binds to and activates torsinA, an AAA+ ATPase that resides in the perinuclear space and continuous main ER. Deletion of torsinA from mouse hepatocytes caused even greater reductions in VLDL secretion and profound steatosis. Both of these mutant mouse lines developed hepatic steatosis and subsequent steatohepatitis on a regular chow diet in the absence of whole-body insulin resistance or obesity. Our results establish an essential role for the nuclear envelope-localized torsinA-LAP1 complex in hepatic VLDL secretion and suggest that the torsinA pathway participates in the pathophysiology of nonalcoholic fatty liver disease.
Ji-Yeon Shin, Antonio Hernandez-Ono, Tatyana Fedotova, Cecilia Östlund, Michael J. Lee, Sarah B. Gibeley, Chun-Chi Liang, William T. Dauer, Henry N. Ginsberg, Howard J. Worman
There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.
Kun Ding, Jikui Shen, Zibran Hafiz, Sean F. Hackett, Raquel Lima e Silva, Mahmood Khan, Valeria E. Lorenc, Daiqin Chen, Rishi Chadha, Minie Zhang, Sherri Van Everen, Nicholas Buss, Michele Fiscella, Olivier Danos, Peter A. Campochiaro
Checkpoint blockade antibodies have been approved as immunotherapy for multiple types of cancer, but the response rate and efficacy are still limited. There are few immunogenic cell death (ICD)-inducing drugs available that can kill cancer cells, enhance tumor immunogenicity, increase the in vivo immune infiltration, and thereby boosting a tumor response to immunotherapy. So far, the ICD markers have been identified as the few immuno-stimulating characteristics of dead cells, but whether the presence of such ICD markers on tumor cells translates into enhanced antitumor immunity in vivo is still investigational. To identify anticancer drugs that could induce tumor cell death and boost T cell response, we performed drug screenings based on both an ICD reporter assay and T cell activation assay. We identified that teniposide, a DNA topoisomerase II inhibitor, could induce high mobility group box 1 (HMGB1) release and type I interferon signaling in tumor cells, and teniposide-treated tumor cells could activate antitumor T cell response both in vitro and in vivo. Mechanistically, teniposide induced tumor cell DNA damage and innate immune signaling including NF-κB activation and STING-dependent type I interferon signaling, both of which contribute to the activation of dendritic cells and subsequent T cells. Furthermore, teniposide potentiated the antitumor efficacy of anti-PD1 on multiple types of mouse tumor models. Our findings showed that teniposide could trigger tumor immunogenicity, and enabled a potential chemo-immunotherapeutic approach to potentiate the therapeutic efficacy of anti-PD1 immunotherapy.
Zining Wang, Jiemin Chen, Jie Hu, Hongxia Zhang, Feifei Xu, Wenzhuo He, Xiaojuan Wang, Mengyun Li, Wenhua Lu, Gucheng Zeng, Penghui Zhou, Peng Huang, Siyu Chen, Wende Li, Liang-ping Xia, Xiaojun Xia
T cell autoreactivity is a hallmark of autoimmune diseases but can also benefit self-maintenance and foster tissue repair. Herein, we investigated whether heart-specific T cells exert salutary or detrimental effects in the context of myocardial infarction (MI), the leading cause of death worldwide. After screening more than 150 class-II-restricted epitopes, we found that myosin heavy chain alpha (MYHCA) was a dominant cardiac antigen triggering post-MI CD4+ T cell activation in mice. Transferred MYHCA614-629-specific CD4+ T (TCR-M) cells selectively accumulated in the myocardium and mediastinal lymph nodes (med-LN) of infarcted mice, acquired a Treg phenotype with a distinct pro-healing gene expression profile, and mediated cardioprotection. Myocardial Treg cells were also detected in autopsies from patients who suffered a MI. Noninvasive PET/CT imaging using a CXCR4 radioligand revealed enlarged med-LNs with increased cellularity in MI-patients. Notably, the med-LN alterations observed in MI patients correlated with the infarct size and cardiac function. Taken together, the results obtained in our study provide evidence showing that MI-context induces pro-healing T cell autoimmunity in mice and confirms the existence of an analogous heart/med-LN/T cell axis in MI patients.
Max Rieckmann, Murilo Delgobo, Chiara Gaal, Lotte Büchner, Philipp Steinau, Dan Reshef, Cristina Gil-Cruz, Ellis N. ter Horst, Malte Kircher, Theresa Reiter, Katrin G. Heinze, Hans W.M. Niessen, Paul A.J. Krijnen, Anja M. van der Laan, Jan J. Piek, Charlotte Koch, Hans-Jürgen Wester, Constantin Lapa, Wolfgang R. Bauer, Burkhard Ludewig, Nir Friedman, Stefan Frantz, Ulrich Hofmann, Gustavo Campos Ramos
A vaccine for hepatitis C virus (HCV) is urgently needed. Development of broadly-neutralizing plasma antibodies during acute infection is associated with HCV clearance, but the viral epitopes of these plasma antibodies are unknown. Identification of these epitopes could define the specificity and function of neutralizing antibodies (NAbs) that should be induced by a vaccine. Here, we present development and application of a high-throughput method that deconvolutes polyclonal anti-HCV NAbs in plasma, delineating the epitope specificities of anti-HCV NAbs in acute infection plasma of forty-four humans with subsequent clearance or persistence of HCV. Remarkably, we identified multiple broadly neutralizing antibody (bNAb) combinations that were associated with greater plasma neutralizing breadth and with HCV clearance. These studies have potential to inform new strategies for vaccine development by identifying bNAb combinations in plasma associated with natural clearance of HCV, while also providing a high-throughput assay that could identify these responses after vaccination trials.
Valerie J. Kinchen, Guido Massaccesi, Andrew I. Flyak, Madeleine C. Mankowski, Michelle D. Colbert, William O. Osburn, Stuart C. Ray, Andrea L. Cox, James E. Crowe Jr., Justin R. Bailey
Serine rich splicing factor 3 (SRSF3) plays a critical role in liver function and its loss promotes chronic liver damage and regeneration. As a consequence, genetic deletion of SRSF3 in hepatocytes caused progressive liver disease and ultimately led to hepatocellular carcinoma. Here we show that SRSF3 is decreased in human liver samples with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis that was associated with alterations in RNA splicing of known SRSF3 target genes. Hepatic SRSF3 expression was similarly decreased and RNA splicing dysregulated in mouse models of NAFLD and NASH. We showed that palmitic acid-induced oxidative stress caused conjugation of the ubiquitin like NEDD8 protein to SRSF3 and proteasome mediated degradation. SRSF3 was selectively neddylated at lysine11 and mutation of this residue (SRSF3-K11R) was sufficient to prevent both SRSF3 degradation and alterations in RNA splicing. Finally prevention of SRSF3 degradation in vivo partially protected mice from hepatic steatosis, fibrosis and inflammation. These results highlight a neddylation-dependent mechanism regulating gene expression in the liver that is disrupted in early metabolic liver disease and may contribute to the progression to NASH, cirrhosis and ultimately hepatocellular carcinoma.
Deepak Kumar, Manasi Das, Consuelo Sauceda, Lesley G. Ellies, Karina Kuo, Purva Parwal, Mehak Kaur, Lily Jih, Gautam K. Bandyopadhyay, Douglas Burton, Rohit Loomba, Olivia Osborn, Nicholas J.G. Webster