Research
Abstract
Secretory protein misfolding has been linked to ER stress and cell death. We expressed a TGrdw transgene encoding TG-G(2298)R, a misfolded mutant thyroglobulin reported to be linked to thyroid cell death. When the TGrdw transgene was expressed at low-level in thyrocytes of TGcog/cog mice that experience severe ER stress, we observed increased thyrocyte cell death and increased expression of CIDE-A (Cell death-inducing DFFA-like effector-A, a protein of lipid droplets) in whole thyroid gland. Here we demonstrate that acute ER stress in cultured PCCL3 thyrocytes increases Cidea mRNA levels, maintained at least in part by increased mRNA stability, while being negatively regulated by ATF6 — with similar observations other cell types. CIDE-A protein is sensitive to proteasomal degradation yet is stabilized by ER stress, and elevated expression levels accompany increased cell death. Unlike acute ER stress, PCCL3 cells adapted and surviving chronic ER stress maintain a disproportionately lower relative mRNA level of Cidea compared to that of other, classical ER stress markers, as well as a blunted Cidea mRNA response to a new, unrelated acute ER stress challenge. We suggest that CIDE-A is a novel marker linked to a non-canonical ER stress-response program, with implications for cell death and survival.
Authors
Yoshiaki Morishita, Aaron P. Kellogg, Dennis Larkin, Wei Chen, Suryakiran Vadrevu, Leslie S. Satin, Ming Liu, Peter Arvan
Abstract
Gene replacement for Duchenne muscular dystrophy (DMD) with micro-dystrophins has entered clinical trials, but efficacy on preventing heart failure is unknown. Although most DMD patients die from heart failure, cardiomyopathy is undetectable until the teens so efficacy from trials in young boys will be unknown for a decade. Available DMD animal models were sufficient to demonstrate micro-dystrophin efficacy on earlier onset skeletal muscle pathology underlying loss of ambulation and respiratory insufficiency in patients. However, no mouse models progressed into heart failure and dog models show highly variable progression insufficient to evaluate efficacy of micro-dystrophin or other therapies on DMD heart failure. To overcome this barrier, we have generated the first DMD mouse model that reproducibly progresses into heart failure. This model shows cardiac inflammation and fibrosis occur prior to reduced function. Fibrosis does not continue to accumulate, but inflammation persists after function declines. We used this model to test micro-dystrophin gene therapy efficacy on heart failure prevention for the first time. Micro-dystrophin prevents declines in cardiac function and prohibits onset of inflammation and fibrosis. This model will allow identification of committed pathogenic steps to heart failure and testing of genetic and non-genetic therapies to optimize cardiac care for DMD patients.
Authors
Zachary M. Howard, Lisa E. Dorn, Jeovanna Lowe, Megan D. Gertzen, Pierce C. Ciccone, Neha Rastogi, Guy L. Odom, Federica Accornero, Jeffrey S. Chamberlain, Jill A. Rafael-Fortney
Abstract
There are approximately 44,000 cases of human papilloma virus (HPV)‒associated cancer each year in the United States, most commonly caused by HPV16/18. Prophylactic vaccines successfully prevent healthy people from acquiring HPV infections via HPV-specific antibodies. To treat established HPV-associated malignancies, however, new therapies are necessary. Multiple recombinant gorilla adenovirus HPV vaccine constructs were evaluated in NSG β2m-/- peripheral blood mononuclear cell–humanized mice bearing SiHa, a human HPV16+ cervical tumor, and/or in the syngeneic HPV16+ TC-1 model. PRGN-2009 is a new therapeutic gorilla adenovirus HPV vaccine containing multiple cytotoxic T-cell epitopes of the viral oncoproteins HPV 16/18 E6 and E7, including T-cell enhancer agonist epitopes. PRGN-2009 treatment reduced tumor volume and increased CD8 and CD4 T cells in the tumor microenvironment of humanized mice bearing the human cervical tumor SiHa. PRGN-2009 monotherapy in the syngeneic TC-1 model also reduced tumor volumes and weights, generated high levels of HPV16 E6-specific T cells, and increased multifunctional CD8 and CD4 T cells in the tumor microenvironment. These studies provide the first evaluation of a therapeutic gorilla adenovirus HPV vaccine, PRGN-2009, showing promising preclinical anti-tumor efficacy and induction of HPV-specific T cells, and the rationale for its evaluation in clinical trials.
Authors
Samuel T. Pellom, Claire Smalley Rumfield, Y. Maurice Morillon II, Nicholas Roller, Lisa K. Poppe, Douglas E. Brough, Helen Sabzevari, Jeffrey Schlom, Caroline Jochems
Abstract
Chronic inflammation and immune dysfunction play a key role in the development of non-AIDS related comorbidities. The aim of our study was to characterize the functional phenotype of immune cells in people living with HIV (PLHIV). We enrolled a cross-sectional cohort study of PLHIV on stable antiretroviral therapy and healthy controls. We assessed ex vivo cytokine production capacity and transcriptomics of monocytes and T-cells upon bacterial, fungal and viral stimulation. PLHIV exhibited an exacerbated pro-inflammatory profile in monocyte-derived cytokines, but not in lymphocyte-derived cytokines. Particularly, the production of the IL-1β to imiquimod, E. coli LPS and Mycobacterium tuberculosis was increased, and this production correlated with plasma concentrations of hsCRP and sCD14. This increase in monocyte responsiveness remained stable over time in subsequent blood sampling after >1year. Transcriptome analyses confirmed priming of the monocyte IL-1β pathway, consistent with a monocyte trained immunity phenotype. Increased plasma concentrations of β-glucan, a well-known inducer of trained immunity, were associated with increased innate cytokine responses. Monocytes of PLHIV exhibit a sustained pro-inflammatory immune phenotype with priming of the IL-1β pathway. Training of the innate immune system in PLHIV likely plays a role in long-term HIV complications and provides a promising therapeutic target for inflammation-related comorbidities.
Authors
Wouter A. van der Heijden, Lisa van de Wijer, Farid Keramati, Wim Trypsteen, Sofie Rutsaert, Rob ter Horst, Martin Jaeger, Hans J.P.M. Koenen, Hendrik G. Stunnenberg, Irma Joosten, Paul E. Verweij, Jan van Lunzen, Charles A. Dinarello, Leo A.B. Joosten, Linos Vandekerckhove, Mihai G. Netea, André J. van der Ven, Quirijn de Mast
Abstract
Primary Graft Dysfunction (PGD) is the predominant cause of early graft loss following lung transplantation. We recently demonstrated that donor pulmonary intravascular non-classical monocytes (NCM) initiate neutrophil recruitment. Simultaneously, host-origin classical monocytes (CM) permeabilize the vascular endothelium to allow neutrophil extravasation necessary for PGD. Here, we show that a CCL2-CCR2 axis is necessary for CM recruitment. Surprisingly, although intravital imaging and multichannel flowcytometry revealed that depletion of donor NCM abrogated CM recruitment, single-cell RNA-seq identified donor alveolar macrophages (AM) as predominant CCL2 secretors. Unbiased transcriptomic analysis of murine tissues combined with murine knockouts and chimeras indicated that IL1β production by donor NCM was responsible for the early activation of AM and CCL2 release. IL1β production by NCM was NLRP3 inflammasome-dependent and inhibited by treatment with a clinically approved sulphonylurea. Production of CCL2 in the donor AM occurred through IL1R-dependent activation of the PKC and NFκB-pathway. Accordingly, we show that IL1β-dependent paracrine interaction between donor NCM and AM leads to recruitment of recipient CM necessary for PGD. Since depletion of donor NCM, IL1β or IL1R antagonism, and inflammasome inhibition, abrogated recruitment of CM as well as PGD, and are feasible using FDA-approved compounds, our findings may have potential for clinical translation.
Authors
Chitaru Kurihara, Emilia Lecuona, Qiang Wu, Wenbin Yang, Felix L. Nunez-Santana, Mahzad Akbarpour, Xianpeng Liu, Ziyou Ren, Wenjun Li, Melissa Querrey, Sowmya Ravi, Megan L. Anderson, Emily Cerier, Haiying Sun, Megan E. Kelly, Hiam Abdala-Valencia, Ali Shilatifard, Thalachallour Mohanakumar, G.R. Scott Budinger, Daniel Kreisel, Ankit Bharat
Abstract
X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich Syndrome protein (WASp). XLN patients have reduced numbers of cytotoxic cells in peripheral blood, however, their capacity to kill tumor cells remains to be determined. Here, we examined NK and T cells from two XLN patients harboring the activating WASpL270P mutation. XLN patient NK and T cells had increased Granzyme B content and elevated degranulation and IFNγ production when compared to healthy control cells. Murine WASpL272P NK and T cells formed stable synapses with YAC-1 tumor cells and anti-CD3/CD28 coated beads, respectively. WASpL272P T cells mice had normal degranulation and cytokine response whereas WASpL272P NK cells showed an enhanced response. Imaging experiments revealed that while WASpL272P CD8 T cells had increased accumulation of actin upon TCR activation, WASpL272P NK cells had normal actin accumulation at lytic synapses triggered through NKp46 signaling but had impaired response to LFA-1 engagement. When compared to WT mice, WASpL272P mice showed reduced growth of B16 melanoma and increased capacity to reject MHC class I-deficient cells. Together, our data suggests that cytotoxic cells with constitutively active WASp have an increased capacity to respond to and kill tumor cells.
Authors
Joanna S. Kritikou, Mariana M.S. Oliveira, Julien Record, Mezida B. Saeed, Saket M. Nigam, Minghui He, Marton Keszei, Arnika K. Wagner, Hanna Brauner, Anton Sendel, Saikiran K. Sedimbi, Stamatina Rentouli, David P. Lane, Scott B. Snapper, Klas Kärre, Peter Vandenberghe, Jordan S. Orange, Lisa S. Westerberg
Abstract
Studies of human hepatitis B virus (HBV) immune pathogenesis are hampered by limited access to liver tissues and technologies for detailed analyses. Here, utilizing imaging mass cytometry (IMC) to simultaneously detect 30 immune, viral and structural markers in liver biopsies from patients with HBeAg+ chronic hepatitis B, we provide novel comprehensive visualization, quantitation and phenotypic characterizations of hepatic adaptive and innate immune subsets that correlated with hepatocellular injury, histological fibrosis and age. We further show marked correlations between adaptive and innate immune cell frequencies and phenotype, highlighting complex immune interactions within the hepatic microenvironment with relevance to HBV pathogenesis.
Authors
Daniel Traum, Yue J. Wang, Kathleen B. Schwarz, Jonathan Schug, David K.H. Wong, Harry L.A. Janssen, Norah A. Terrault, Mandana Khalili, Abdus S. Wahed, Karen F. Murray, Philip Rosenthal, Simon C. Ling, Norberto Rodriguez-Baez, Richard K. Sterling, Daryl T.Y. Lau, Timothy M. Block, Michael D. Feldman, Emma E. Furth, William M. Lee, David E. Kleiner, Anna S. Lok, Klaus H. Kaestner, Kyong-Mi Chang
Abstract
Comorbid medical illnesses, such as obesity and diabetes, are associated with more severe COVID-19, hospitalization, and death. However, the role of the immune system in mediating these clinical outcomes has not been determined. We used multi-parameter flow cytometry and systems serology to comprehensively profile the functions of T cells and antibodies targeting spike, nucleocapsid, and envelope proteins in a convalescent cohort of COVID-19 subjects who were either hospitalized (n=20) or not hospitalized (n=40). To avoid confounding, subjects were matched by age, sex, ethnicity, and date of symptom onset. Surprisingly, we found that the magnitude and functional breadth of virus-specific CD4 T cell and antibody responses were consistently higher among hospitalized subjects, particularly those with medical comorbidities. However, an integrated analysis identified more coordination between polyfunctional CD4 T-cells and antibodies targeting the S1 domain of spike among subjects that were not hospitalized. These data reveal a functionally diverse and coordinated response between T cells and antibodies targeting SARS-CoV-2, which is reduced in the presence of comorbid illnesses that are known risk factors for severe COVID-19.
Authors
Krystle K.Q. Yu, Stephanie Fischinger, Malisa T. Smith, Caroline Atyeo, Deniz Cizmeci, Caitlin R. Wolf, Erik D. Layton, Jennifer K. Logue, Melissa S. Aguilar, Kiel Shuey, Carolin Loos, Jingyou Yu, Nicholas M. Franko, Robert Y. Choi, Anna Wald, Dan H. Barouch, David M. Koelle, Douglas Lauffenburger, Helen Y. Chu, Galit Alter, Chetan Seshadri
Abstract
The drive to withstand environmental stresses and defend against invasion is a universal trait extant in all forms of life. While numerous canonical signaling cascades have been characterized in detail, it remains unclear how these pathways interface to generate coordinated responses to diverse stimuli. To dissect these connections, we follow heparanase (HPSE), a protein best known for its endoglycosidic activity at the extracellular matrix but recently recognized to drive various forms of late stage disease through unknown mechanisms. Using herpes simplex virus-1 (HSV-1) infection as a model cellular perturbation, we demonstrate that HPSE acts beyond its established enzymatic role to restrict multiple forms of cell-intrinsic defense and facilitate host cell reprogramming by the invading pathogen. We reveal that cells devoid of HPSE are innately resistant to infection and counteract viral takeover through multiple amplified defense mechanisms. With a unique grasp of the fundamental processes of transcriptional regulation and cell death, HPSE represents a potent cellular intersection with broad therapeutic potential.
Authors
Alex Agelidis, Benjamin A. Turturice, Rahul K. Suryawanshi, Tejabhiram Yadavalli, Dinesh Jaishankar, Joshua Ames, James Hopkins, Lulia Koujah, Chandrashekhar D. Patil, Satvik R. Hadigal, Evan J. Kyzar, Anaamika Campeau, Jacob M. Wozniak, David J. Gonzalez, Israel Vlodavsky, Jin-ping Li, David L. Perkins, Patricia W. Finn, Deepak Shukla
Abstract
The splenic microenvironment regulates hematopoietic stem and progenitor cell (HSPC) function, particularly during demand-adapted hematopoiesis, however practical strategies to enhance splenic support of transplanted HSPCs have proven elusive. We have previously demonstrated that inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH), using the small molecule (+)SW033291 (PGDHi), increases bone marrow (BM) prostaglandin E2 (PGE2) levels, expands HSPC numbers, and accelerates hematologic reconstitution following BM transplantation (BMT) in mice. Here we demonstrate that the splenic microenvironment, specifically 15-PGDH high-expressing macrophages (MΦs), megakaryocytes (MKs), and mast cells (MCs), regulates steady-state hematopoiesis and potentiates recovery after BMT. Notably, PGDHi-induced neutrophil, platelet, and HSPC recovery were highly attenuated in splenectomized mice. PGDHi induced non-pathologic splenic extramedullary hematopoiesis at steady-state, and pre-transplant PGDHi enhanced the homing of transplanted cells to the spleen. 15-PGDH enzymatic activity localized specifically to MΦs, MK lineage cells, and MCs, identifying these cell types as likely coordinating the impact of PGDHi on splenic HSPCs. These findings suggest that 15-PGDH expression marks novel HSC niche cell types that regulate hematopoietic regeneration. Therefore, PGDHi provides a well-tolerated strategy to therapeutically target multiple HSC niches and to promote hematopoietic regeneration and improve clinical outcomes of BMT.
Authors
Julianne N.P. Smith, Dawn M. Dawson, Kelsey F. Christo, Alvin P. Jogasuria, Mark J. Cameron, Monika I. Antczak, Joseph M. Ready, Stanton L. Gerson, Sanford D. Markowitz, Amar B. Desai
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