Triple-negative breast cancers (TNBCs) lack effective targeted therapies, and cytotoxic chemotherapies remain the standard of care for this subtype. Owing to their increased genomic instability, poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) are being tested against TNBCs. In particular, clinical trials are now interrogating the efficacy of PARPi combined with chemotherapies. Intriguingly, while response rates are low, cohort of patients do respond to PARPi in combination with chemotherapies. Moreover, recent studies suggest that an increase in levels of ROS may sensitize cells to PARPi. This represents a therapeutic opportunity, as several chemotherapies, including doxorubicin, function in part by producing ROS. We previously demonstrated that the p66ShcA adaptor protein is variably expressed in TNBCs. We now show that, in response to therapy-induced stress, p66ShcA stimulated ROS production, which, in turn, potentiated the synergy of PARPi in combination with doxorubicin in TNBCs. This p66ShcA-induced sensitivity relied on the accumulation of oxidative damage in TNBCs, rather than genomic instability, to potentiate cell death. These findings suggest that increasing the expression of p66ShcA protein levels in TNBCs represents a rational approach to bolster the synergy between PARPi and doxorubicin.
Eduardo Cepeda Cañedo, Stephanie Totten, Ryuhjin Ahn, Paul Savage, Deanna MacNeil, Jesse Hudson, Chantal Autexier, Genevieve Deblois, Morag Park, Michael Witcher, Josie Ursini-Siegel
Background Mitochondrial DNA (MT-DNA) are intrinsically inflammatory nucleic acids released by damaged solid organs. Whether circulating cell-free MT-DNA quantitation could be used to predict the risk of poor COVID-19 outcomes remains undetermined.Methods We measured circulating MT-DNA levels in prospectively collected, cell-free plasma samples from 97 subjects with COVID-19 at hospital presentation. Our primary outcome was mortality. Intensive care unit (ICU) admission, intubation, vasopressor, and renal replacement therapy requirements were secondary outcomes. Multivariate regression analysis determined whether MT-DNA levels were independent of other reported COVID-19 risk factors. Receiver operating characteristic and area under the curve assessments were used to compare MT-DNA levels with established and emerging inflammatory markers of COVID-19.Results Circulating MT-DNA levels were highly elevated in patients who eventually died or required ICU admission, intubation, vasopressor use, or renal replacement therapy. Multivariate regression revealed that high circulating MT-DNA was an independent risk factor for these outcomes after adjusting for age, sex, and comorbidities. We also found that circulating MT-DNA levels had a similar or superior area under the curve when compared against clinically established measures of inflammation and emerging markers currently of interest as investigational targets for COVID-19 therapy.Conclusion These results show that high circulating MT-DNA levels are a potential early indicator for poor COVID-19 outcomes.Funding Washington University Institute of Clinical Translational Sciences COVID-19 Research Program and Washington University Institute of Clinical Translational Sciences (ICTS) NIH grant UL1TR002345.
Davide Scozzi, Marlene Cano, Lina Ma, Dequan Zhou, Ji Hong Zhu, Jane A. O’Halloran, Charles Goss, Adriana M. Rauseo, Zhiyi Liu, Sanjaya K. Sahu, Valentina Peritore, Monica Rocco, Alberto Ricci, Rachele Amodeo, Laura Aimati, Mohsen Ibrahim, Ramsey Hachem, Daniel Kreisel, Philip A. Mudd, Hrishikesh S. Kulkarni, Andrew E. Gelman
Somatostatin (SS) inhibits glucagon-like peptide-1 (GLP-1) secretion in a paracrine manner. We hypothesized that blocking somatostatin subtype receptor 2 (SSTR2) and 5 (SSTR5) would improve glycemia by enhancing GLP-1 secretion. In the perfused mouse small intestine, the selective SSTR5 antagonist (SSTR5a) stimulated glucose-induced GLP-1 secretion to a larger degree than the SSTR2 antagonist (SSTR2a). In parallel, mice lacking the SSTR5R showed increased glucose-induced GLP-1 secretion. Both antagonists improved glycemia in vivo in a GLP-1 receptor–dependent (GLP-1R–dependent) manner, as the glycemic improvements were absent in mice with impaired GLP-1R signaling and in mice treated with a GLP-1R–specific antagonist. SSTR5a had no direct effect on insulin secretion in the perfused pancreas, whereas SSTR2a increased insulin secretion in a GLP-1R–independent manner. Adding a dipeptidyl peptidase 4 inhibitor (DPP-4i) in vivo resulted in additive effects on glycemia. However, when glucose was administered intraperitoneally, the antagonist was incapable of lowering blood glucose. Oral administration of SSTR5a, but not SSTR2a, lowered blood glucose in diet-induced obese mice. In summary, we demonstrate that selective SSTR antagonists can improve glucose control primarily through the intestinal GLP-1 system in mice.
Sara L. Jepsen, Nicolai J. Wewer Albrechtsen, Johanne A. Windeløv, Katrine D. Galsgaard, Jenna E. Hunt, Thomas B. Farb, Hannelouise Kissow, Jens Pedersen, Carolyn F. Deacon, Rainer E. Martin, Jens J. Holst
Glucagon regulates glucose and lipid metabolism and promotes weight loss. Thus, therapeutics stimulating glucagon receptor (GCGR) signaling are promising for obesity treatment; however, the underlying mechanism(s) have yet to be fully elucidated. We previously identified that hepatic GCGR signaling increases circulating fibroblast growth factor 21 (FGF21), a potent regulator of energy balance. We reported that mice deficient for liver Fgf21 are partially resistant to GCGR-mediated weight loss, implicating FGF21 as a regulator of glucagon’s weight loss effects. FGF21 signaling requires an obligate coreceptor (β-Klotho, KLB), with expression limited to adipose tissue, liver, pancreas, and brain. We hypothesized that the GCGR-FGF21 system mediates weight loss through a central mechanism. Mice deficient for neuronal Klb exhibited a partial reduction in body weight with chronic GCGR agonism (via IUB288) compared with controls, supporting a role for central FGF21 signaling in GCGR-mediated weight loss. Substantiating these results, mice with central KLB inhibition via a pharmacological KLB antagonist, 1153, also displayed partial weight loss. Central KLB, however, is dispensable for GCGR-mediated improvements in plasma cholesterol and liver triglycerides. Together, these data suggest GCGR agonism mediates part of its weight loss properties through central KLB and has implications for future treatments of obesity and metabolic syndrome.
Shelly R. Nason, Jessica Antipenko, Natalie Presedo, Stephen E. Cunningham, Tanya H. Pierre, Teayoun Kim, Jodi R. Paul, Cassie Holleman, Martin E. Young, Karen L. Gamble, Brian Finan, Richard DiMarchi, Chad S. Hunter, Alexei Kharitonenkov, Kirk M. Habegger
Convalescent plasma with severe acute respiratory disease coronavirus 2 (SARS-CoV-2) antibodies (CCP) may hold promise as a treatment for coronavirus disease 2019 (COVID-19). We compared the mortality and clinical outcome of patients with COVID-19 who received 200 mL of CCP with a spike protein IgG titer ≥ 1:2430 (median 1:47,385) within 72 hours of admission with propensity score–matched controls cared for at a medical center in the Bronx, between April 13 and May 4, 2020. Matching criteria for controls were age, sex, body mass index, race, ethnicity, comorbidities, week of admission, oxygen requirement, D-dimer, lymphocyte counts, corticosteroid use, and anticoagulation use. There was no difference in mortality or oxygenation between CCP recipients and controls at day 28. When stratified by age, compared with matched controls, CCP recipients less than 65 years had 4-fold lower risk of mortality and 4-fold lower risk of deterioration in oxygenation or mortality at day 28. For CCP recipients, pretransfusion spike protein IgG, IgM, and IgA titers were associated with mortality at day 28 in univariate analyses. No adverse effects of CCP were observed. Our results suggest CCP may be beneficial for hospitalized patients less than 65 years, but data from controlled trials are needed to validate this finding and establish the effect of aging on CCP efficacy.
Hyun ah Yoon, Rachel Bartash, Inessa Gendlina, Johanna Rivera, Antonio Nakouzi, Robert H. Bortz III, Ariel S. Wirchnianski, Monika Paroder, Karen Fehn, Leana Serrano-Rahman, Rachelle Babb, Uzma N. Sarwar, Denise Haslwanter, Ethan Laudermilch, Catalina Florez, M. Eugenia Dieterle, Rohit K. Jangra, J. Maximilian Fels, Karen Tong, Margarette C. Mariano, Olivia Vergnolle, George I. Georgiev, Natalia G. Herrera, Ryan J. Malonis, Jose A. Quiroz, Nicholas C. Morano, Gregory J. Krause, Joseph M. Sweeney, Kelsie Cowman, Stephanie Allen, Jayabhargav Annam, Ariella Applebaum, Daniel Barboto, Ahmed Khokhar, Brianna J. Lally, Audrey Lee, Max Lee, Avinash Malaviya, Reise Sample, Xiuyi A. Yang, Yang Li, Rafael Ruiz, Raja Thota, Jason Barnhill, Doctor Y. Goldstein, Joan Uehlinger, Scott J. Garforth, Steven C. Almo, Jonathan R. Lai, Morayma Reyes Gil, Amy S. Fox, Kartik Chandran, Tao Wang, Johanna P. Daily, Liise-anne Pirofski
MC4R mutations represent the largest monogenic cause of obesity, resulting mainly from receptor misfolding and intracellular retention by the cellular quality control system. The present study aimed at determining whether pharmacological chaperones (PCs) that restore folding and plasma membrane trafficking by stabilizing near native protein conformation may represent valid therapeutic avenues for the treatment of melanocortin type 4 receptor–linked (MC4R-linked) obesity. To test the therapeutic PC potential, we engineered humanized MC4R (hMC4R) mouse models expressing either the WT human MC4R or a prevalent obesity-causing mutant (R165W). Administration of a PC able to rescue cell surface expression and functional activity of R165W-hMC4R in cells restored the anorexigenic response of the R165W-hMC4R obese mice to melanocortin agonist, providing a proof of principle for the therapeutic potential of MC4R-targeting PCs in vivo. Interestingly, the expression of the WT-hMC4R in mice revealed lower sensitivity of the human receptor to α–melanocyte-stimulating hormone (α-MSH) but not β-MSH or melanotan II, resulting in a lower penetrance obese phenotype in the WT-hMC4R versus R165W-hMC4R mice. In conclusion, we created 2 new obesity models, a hypomorphic highlighting species differences and an amorphic providing a preclinical model to test the therapeutic potential of PCs to treat MC4R-linked obesity.
Patricia René, Damien Lanfray, Denis Richard, Michel Bouvier
Four endemic human coronaviruses (HCoVs) are commonly associated with acute respiratory infection in humans. B cell responses to these “common cold” viruses remain incompletely understood. Here we report a comprehensive analysis of CoV-specific antibody repertoires in 231 children and 1168 adults using phage immunoprecipitation sequencing. Seroprevalence of antibodies against endemic HCoVs ranged between approximately 4% and 27% depending on the species and cohort. We identified at least 136 novel linear B cell epitopes. Antibody repertoires against endemic HCoVs were qualitatively different between children and adults in that anti-HCoV IgG specificities more frequently found among children targeted functionally important and structurally conserved regions of the spike, nucleocapsid, and matrix proteins. Moreover, antibody specificities targeting the highly conserved fusion peptide region and S2′ cleavage site of the spike protein were broadly cross-reactive with peptides of epidemic human and nonhuman coronaviruses. In contrast, an acidic tandem repeat in the N-terminal region of the Nsp3 subdomain of the HCoV-HKU1 polyprotein was the predominant target of antibody responses in adult donors. Our findings shed light on the dominant species-specific and pan-CoV target sites of human antibody responses to coronavirus infection, thereby providing important insights for the development of prophylactic or therapeutic monoclonal antibodies and vaccine design.
Taushif Khan, Mahbuba Rahman, Fatima Al Ali, Susie S. Y. Huang, Manar Ata, Qian Zhang, Paul Bastard, Zhiyong Liu, Emmanuelle Jouanguy, Vivien Béziat, Aurélie Cobat, Gheyath K. Nasrallah, Hadi M. Yassine, Maria K. Smatti, Amira Saeed, Isabelle Vandernoot, Jean-Christophe Goffard, Guillaume Smits, Isabelle Migeotte, Filomeen Haerynck, Isabelle Meyts, Laurent Abel, Jean-Laurent Casanova, Mohammad R. Hasan, Nico Marr
Diarrhea is a major cause of global mortality, and outbreaks of secretory diarrhea such as cholera remain an important problem in the developing world. Current treatment of secretory diarrhea primarily involves supportive measures, such as fluid replacement. The calcium-sensing receptor (CaSR) regulates multiple biological activities in response to changes in extracellular Ca2+. The FDA-approved drug cinacalcet is an allosteric activator of CaSR used for treatment of hyperparathyroidism. Here, we found by short-circuit current measurements in human colonic T84 cells that CaSR activation by cinacalcet reduced forskolin-induced Cl– secretion by greater than 80%. Cinacalcet also reduced Cl– secretion induced by cholera toxin, heat-stable E. coli enterotoxin, and vasoactive intestinal peptide (VIP). The cinacalcet effect primarily involved indirect inhibition of cystic fibrosis transmembrane conductance regulator–mediated (CFTR-mediated) Cl– secretion following activation of CaSR and downstream phospholipase C and phosphodiesterases. In mice, cinacalcet reduced fluid accumulation by more than 60% in intestinal closed loop models of cholera and traveler’s diarrhea. The cinacalcet effect involved both inhibition of CFTR-mediated secretion and stimulation of sodium-hydrogen exchanger 3–mediated absorption. These findings support the therapeutic utility of the safe and commonly used drug cinacalcet in CFTR-dependent secretory diarrheas, including cholera, traveler’s diarrhea, and VIPoma.
Apurva A. Oak, Parth D. Chhetri, Amber A. Rivera, Alan S. Verkman, Onur Cil
Multiple myeloma (MM) is characterized by an accumulation of malignant plasma cells (PCs) within the BM. The BM microenvironment supports survival of the malignant cells and is composed of cellular fractions that foster myeloma development and progression by suppression of the immune response. Despite major progress in understanding the biology and pathophysiology of MM, this disease is still incurable and requires aggressive treatment with significant side effects. CD84 is a self-binding immunoreceptor belonging to the signaling lymphocyte activation molecule (SLAM) family. Previously, we showed that CD84 bridges between chronic lymphocytic leukemia cells and their microenvironment, and it regulates T cell function. In the current study, we investigated the role of CD84 in MM. Our results show that MM cells express low levels of CD84. However, these cells secrete the cytokine macrophage migration inhibitory factor (MIF), which induces CD84 expression on cells in their microenvironment. Its activation leads to an elevation of expression of genes regulating differentiation to monocytic/granulocytic–myeloid-derived suppressor cells (M-MDSCs and G-MDSCs, respectively) and upregulation of PD-L1 expression on MDSCs, which together suppress T cell function. Downregulation of CD84 or its blocking reduce MDSC accumulation, resulting in elevated T cell activity and reduced tumor load. Our data suggest that CD84 might serve as a novel therapeutic target in MM.
Hadas Lewinsky, Emine G. Gunes, Keren David, Lihi Radomir, Matthias P. Kramer, Bianca Pellegrino, Michal Perpinial, Jing Chen, Ting-fang He, Anthony G. Mansour, Kun-Yu Teng, Supriyo Bhattacharya, Enrico Caserta, Estelle Troadec, Peter Lee, Mingye Feng, Jonathan Keats, Amrita Krishnan, Michael Rosenzweig, Jianhua Yu, Michael A. Caligiuri, Yosef Cohen, Olga Shevetz, Shirly Becker-Herman, Flavia Pichiorri, Steven Rosen, Idit Shachar
Influenza virus infections affect millions of people annually, and current available vaccines provide varying rates of protection. However, the way in which the nasal microbiota, particularly established pneumococcal colonization, shape the response to influenza vaccination is not yet fully understood. In this study, we inoculated healthy adults with live Streptococcus pneumoniae and vaccinated them 3 days later with either tetravalent-inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV). Vaccine-induced immune responses were assessed in nose, blood, and lung. Nasal pneumococcal colonization had no impact upon TIV-induced antibody responses to influenza, which manifested in all compartments. However, experimentally induced pneumococcal colonization dampened LAIV-mediated mucosal antibody responses, primarily IgA in the nose and IgG in the lung. Pulmonary influenza-specific cellular responses were more apparent in the LAIV group compared with either the TIV or an unvaccinated group. These results indicate that TIV and LAIV elicit differential immunity to adults and that LAIV immunogenicity is diminished by the nasal presence of S. pneumoniae. Therefore, nasopharyngeal pneumococcal colonization may affect LAIV efficacy.
Beatriz F. Carniel, Fernando Marcon, Jamie Rylance, Esther L. German, Seher Zaidi, Jesus Reiné, Edessa Negera, Elissavet Nikolaou, Sherin Pojar, Carla Solórzano, Andrea M. Collins, Victoria Connor, Debbie Bogaert, Stephen B. Gordon, Helder I. Nakaya, Daniela M. Ferreira, Simon P. Jochems, Elena Mitsi
IL-33 is a key mediator of chronic airway disease driven by type 2 immune pathways, yet the nonclassical secretory mechanism for this cytokine remains undefined. We performed a comprehensive analysis in human airway epithelial cells, which revealed that tonic IL-33 secretion is dependent on the ceramide biosynthetic enzyme neutral sphingomyelinase 2 (nSMase2). IL-33 is cosecreted with exosomes by the nSMase2-regulated multivesicular endosome (MVE) pathway as surface-bound cargo. In support of these findings, human chronic obstructive pulmonary disease (COPD) specimens exhibited increased epithelial expression of the abundantly secreted IL33Δ34 isoform and augmented nSMase2 expression compared with non-COPD specimens. Using an Alternaria-induced airway disease model, we found that the nSMase2 inhibitor GW4869 abrogated both IL-33 and exosome secretion as well as downstream inflammatory pathways. This work elucidates a potentially novel aspect of IL-33 biology that may be targeted for therapeutic benefit in chronic airway diseases driven by type 2 inflammation.
Ella Katz-Kiriakos, Deborah F. Steinberg, Colin E. Kluender, Omar A. Osorio, Catie Newsom-Stewart, Arjun Baronia, Derek E. Byers, Michael J. Holtzman, Dawn Katafiasz, Kristina L. Bailey, Steven L. Brody, Mark J. Miller, Jennifer Alexander-Brett
Reestablishing an appropriate balance between T effector cells (Teff) and Tregs is essential for correcting autoimmunity. Multiple sclerosis (MS) is an immune-mediated chronic CNS disease characterized by neuroinflammation, demyelination, and neuronal degeneration, in which the Teff:Treg balance is skewed toward pathogenic Teffs Th1 and Th17 cells. STAT3 is a key regulator of Teff:Treg balance. Using the structure-based design, we have developed a potentially novel small-molecule prodrug LLL12b that specifically inhibits STAT3 and suppresses Th17 differentiation and expansion. Moreover, LLL12b regulates the fate decision between Th17 and Tregs in an inflammatory environment, shifting Th17:Treg balance toward Tregs and favoring the resolution of inflammation. Therapeutic administration of LLL12b after disease onset significantly suppresses disease progression in adoptively transferred, chronic, and relapsing-remitting experimental autoimmune encephalomyelitis. Disease relapses were also significantly suppressed by LLL12b given during the remission phase. Additionally, LLL12b shifts Th17:Treg balance of CD4+ T cells from MS patients toward Tregs and increases Teff sensitivity to Treg-mediated suppression. These data suggest that selective inhibition of STAT3 by the small molecule LLL12b recalibrates the effector and regulatory arms of CD4+ T responses, representing a potentially clinically translatable therapeutic strategy for MS.
Saba I. Aqel, Xiaozhi Yang, Emma E. Kraus, Jinhua Song, Marissa F. Farinas, Erin Y. Zhao, Wei Pei, Amy E. Lovett-Racke, Michael K. Racke, Chenglong Li, Yuhong Yang
The metabotropic glutamate receptor 7 (mGlu7) is a G protein–coupled receptor that has been recently linked to neurodevelopmental disorders. This association is supported by the identification of GRM7 variants in patients with autism spectrum disorder, attention deficit hyperactivity disorder, and severe developmental delay. One GRM7 mutation previously reported in 2 patients results in a single amino acid change, I154T, within the mGlu7 ligand-binding domain. Here, we report 2 new patients with this mutation who present with severe developmental delay and epilepsy. Functional studies of the mGlu7-I154T mutant reveal that this substitution resulted in significant loss of mGlu7 protein expression in HEK293A cells and in mice. We show that this occurred posttranscriptionally at the level of protein expression and trafficking. Similar to mGlu7–global KO mice, mGlu7-I154T animals exhibited reduced motor coordination, deficits in contextual fear learning, and seizures. This provides functional evidence that a disease-associated mutation affecting the mGlu7 receptor was sufficient to cause neurological dysfunction in mice and further validates GRM7 as a disease-causing gene in the human population.
Nicole M. Fisher, Aqeela AlHashim, Aditi B. Buch, Hana Badivuku, Manar M. Samman, Kelly M. Weiss, Gabriela I. Cestero, Mark D. Does, Jerri M. Rook, Craig W. Lindsley, P. Jeffrey Conn, Rocco G. Gogliotti, Colleen M. Niswender
Prime-boost immunization strategies are required to control the global tuberculosis (TB) pandemic, which claims approximately 3 lives every minute. Here, we have generated an immunogenic complex against Mycobacterium tuberculosis (M.tb), consisting of promiscuous T cell epitopes (M.tb peptides) and TLR ligands assembled in liposomes. Interestingly, this complex (peptide–TLR agonist–liposomes; PTL) induced significant activation of CD4+ T cells and IFN-γ production in the PBMCs derived from PPD+ healthy individuals as compared with PPD– controls. Furthermore, intranasal delivery of PTL significantly reduced the bacterial burden in the infected mice by inducing M.tb-specific polyfunctional (IFN-γ+IL-17+TNF-α+IL-2+) immune responses and long-lasting central memory responses, thereby reducing the risk of TB recurrence in DOTS-treated infected animals. The transcriptome analysis of peptide-stimulated immune cells unveiled the molecular basis of enhanced protection. Furthermore, PTL immunization significantly boosted the Bacillus Calmette-Guerin–primed (BCG-primed) immune responses against TB. The greatly enhanced efficacy of the BCG-PTL vaccine model in controlling pulmonary TB projects PTL as an adjunct vaccine against TB.
Santosh Kumar, Ashima Bhaskar, Gautam Patnaik, Chetan Sharma, Dhiraj Kumar Singh, Sandeep Rai Kaushik, Shivam Chaturvedi, Gobardhan Das, Ved Prakash Dwivedi
Most patients with glioblastoma (GBM) die within 2 years. A major therapeutic goal is to target GBM stem cells (GSCs), a subpopulation of cells that contribute to treatment resistance and recurrence. Since their discovery in 2003, GSCs have been isolated using single-surface markers, such as CD15, CD44, CD133, and α6 integrin. It remains unknown how these single-surface marker–defined GSC populations compare with each other in terms of signaling and function and whether expression of different combinations of these markers is associated with different functional capacity. Using mass cytometry and fresh operating room specimens, we found 15 distinct GSC subpopulations in patients, and they differed in their MEK/ERK, WNT, and AKT pathway activation status. Once in culture, some subpopulations were lost and previously undetectable ones materialized. GSCs that highly expressed all 4 surface markers had the greatest self-renewal capacity, WNT inhibitor sensitivity, and in vivo tumorigenicity. This work highlights the potential signaling and phenotypic diversity of GSCs. Larger patient sample sizes and antibody panels are required to confirm these findings.
Luciano Galdieri, Arijita Jash, Olga Malkova, Diane D. Mao, Patrick DeSouza, Yunli E. Chu, Amber Salter, Jian L. Campian, Kristen M. Naegle, Cameron W. Brennan, Hiroaki Wakimoto, Stephen T. Oh, Albert H. Kim, Milan G. Chheda
White adipose tissue (WAT) insulin action has critical anabolic function and is dysregulated in overnutrition. However, the mechanism of short-term high-fat diet–induced (HFD-induced) WAT insulin resistance (IR) is poorly understood. Based on recent evidences, we hypothesize that a short-term HFD causes WAT IR through plasma membrane (PM) sn-1,2-diacylglycerol (sn-1,2-DAG) accumulation, which promotes protein kinase C-ε (PKCε) activation to impair insulin signaling by phosphorylating insulin receptor (Insr) Thr1160. To test this hypothesis, we assessed WAT insulin action in 7-day HFD–fed versus regular chow diet–fed rats during a hyperinsulinemic-euglycemic clamp. HFD feeding caused WAT IR, reflected by impaired insulin-mediated WAT glucose uptake and lipolysis suppression. These changes were specifically associated with PM sn-1,2-DAG accumulation, higher PKCε activation, and impaired insulin-stimulated Insr Tyr1162 phosphorylation. In order to examine the role of Insr Thr1160 phosphorylation in mediating lipid-induced WAT IR, we examined these same parameters in InsrT1150A mice (mouse homolog for human Thr1160) and found that HFD feeding induced WAT IR in WT control mice but not in InsrT1150A mice. Taken together, these data demonstrate the importance of the PM sn-1,2-DAG/PKCε/Insr Thr1160 phosphorylation pathway in mediating lipid-induced WAT IR and represent a potential therapeutic target to improve WAT insulin sensitivity.
Kun Lyu, Dongyan Zhang, Joongyu Song, Xiruo Li, Rachel J. Perry, Varman T. Samuel, Gerald I. Shulman
Rewiring tumor cells to undergo drug-induced apoptosis is a promising way to overcome chemoresistance. Therefore, identifying causative factors for chemoresistance is of high importance. Unbiased global proteome profiling of sensitive, early, and late cisplatin-resistant oral squamous cell carcinoma (OSCC) lines identified CMTM6 as a top-ranked upregulated protein. Analyses of OSCC patient tumor samples demonstrated significantly higher CMTM6 expression in chemotherapy (CT) nonresponders as compared with CT responders. In addition, a significant association between higher CMTM6 expression and poorer relapse-free survival in esophageal squamous cell carcinoma, head and neck squamous cell carcinoma, and lung squamous cell carcinoma was observed from Kaplan-Meier plot analysis. Stable knockdown (KD) of CMTM6 restored cisplatin-mediated cell death in chemoresistant OSCC lines. Upon CMTM6 overexpression in CMTM6-KD lines, the cisplatin-resistant phenotype was rescued. The patient-derived cell xenograft model of chemoresistant OSCC displaying CMTM6 depletion restored the cisplatin-induced cell death and tumor burden substantially. The transcriptome analysis of CMTM6-KD and control chemoresistant cells depicted enrichment of the Wnt signaling pathway. We demonstrated that CMTM6 interaction with membrane-bound Enolase-1 stabilized its expression, leading to activation of Wnt signaling mediated by AKT–glycogen synthase kinase-3β. CMTM6 has been identified as a stabilizer of programmed cell death ligand 1. Therefore, as CMTM6 facilitates tumor cells for immune evasion and mediates cisplatin resistance, it could be a promising therapeutic target for treating therapy-resistant OSCC.
Pallavi Mohapatra, Omprakash Shriwas, Sibasish Mohanty, Arup Ghosh, Shuchi Smita, Sandeep Rai Kaushik, Rakesh Arya, Rachna Rath, Saroj Kumar Das Majumdar, Dillip Kumar Muduly, Sunil K. Raghav, Ranjan K. Nanda, Rupesh Dash
An intact lung epithelial barrier is essential for lung homeostasis. The Na+, K+-ATPase (NKA), primarily serving as an ion transporter, also regulates epithelial barrier function via modulation of tight junctions. However, the underlying mechanism is not well understood. Here, we show that overexpression of the NKA β1 subunit upregulates the expression of tight junction proteins, leading to increased alveolar epithelial barrier function by an ion transport–independent mechanism. Using IP and mass spectrometry, we identified a number of unknown protein interactions of the β1 subunit, including a top candidate, myotonic dystrophy kinase–related cdc42-binding kinase α (MRCKα), which is a protein kinase known to regulate peripheral actin formation. Using a doxycycline-inducible gene expression system, we demonstrated that MRCKα and its downstream activation of myosin light chain is required for the regulation of alveolar barrier function by the NKA β1 subunit. Importantly, MRCKα is expressed in both human airways and alveoli and has reduced expression in patients with acute respiratory distress syndrome (ARDS), a lung illness that can be caused by multiple direct and indirect insults, including the infection of influenza virus and SARS-CoV-2. Our results have elucidated a potentially novel mechanism by which NKA regulates epithelial tight junctions and have identified potential drug targets for treating ARDS and other pulmonary diseases that are caused by barrier dysfunction.
Haiqing Bai, Rui Zhou, Michael Barravecchia, Rosemary Norman, Alan Friedman, Deborah Yu, Xin Lin, Jennifer L. Young, David A. Dean
The coronavirus disease 19 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become the worst public health crisis in a century. However, knowledge about the dynamics of antibody responses in patients with COVID-19 is still poorly understood. In this study, we performed a serological study with serum specimens collected at the acute and the convalescent phases from 104 patients with severe COVID-19 who were part of the first wave of COVID-19 cases in Wuhan, China. Our findings revealed that neutralizing antibodies to SARS-CoV-2 are persistent for at least 6 months in patients with severe COVID-19, despite that IgG levels against the receptor binding domain (RBD) and nucleocapsid protein (N) IgG declined from the acute to the convalescent phase. Moreover, we demonstrate that the level of RBD-IgG is capable of correlating with SARS-CoV-2–neutralizing activities in COVID-19 serum. In summary, our findings identify the magnitude, functionality, and longevity of antibody responses in patients with COVID-19, which sheds light on the humoral immune response to COVID-19 and would be beneficial for developing vaccines.
Yang Han, Peipei Liu, Yang Qiu, Jie Zhou, Ying Liu, Xujuan Hu, Qingyu Yang, Rui Huang, Xinyue Wen, Hao Song, Pengcheng Yu, Mengjie Yang, Jing Zhang, William J. Liu, Ke Peng, Guizhen Wu, Dingyu Zhang, Xi Zhou, Ying Wu
John M. Carethers
Introduction: Coronavirus 2019 (COVID-19) clinical course is heterogeneous, ranging from mild to severe multi-organ failure and death. In this study, we analyzed cell-free DNA (cfDNA) as a biomarker of injury to define the sources of tissue injury that contribute to such different trajectories. Methods: We conducted a multi-center prospective cohort study to enroll COVID-19 patients and collect plasma samples. Plasma cfDNA was subject to bisulfite sequencing. A library of tissue-specific DNA methylation signatures was used to analyze sequence reads to quantitate cfDNA from different tissue types. We then determined the correlation of tissue-specific cfDNA measures to COVID-19 outcomes. Similar analyses was performed for healthy controls and a comparator group of patients with respiratory syncytial virus and influenza. Results: We found markedly elevated levels and divergent tissue sources of cfDNA in COVID-19 patients compared to influenza and respiratory syncytial virus patients or healthy controls. The major sources of cfDNA in COVID-19 were hematopoietic cells, vascular endothelium, hepatocyte, adipocyte, kidney, heart and lung. cfDNA levels positively correlated with COVID-19 disease severity, c reactive protein, D-Dimer. cfDNA profile at admission identified patients who subsequently required intensive care or died during hospitalization. Furthermore, the increased cfDNA in COVID-19 patients generates excessive mitochondrial reactive oxygen species (mtROS) in renal tubular cells in a concentration-dependent manner. This mtROS production was inhibited by a toll-like receptor 9 (TLR-9)-specific antagonist. Conclusion cfDNA maps tissue injury that predict COVID-19 outcomes, and may mechanistically propagates COVID-19 induced tissue injury. Funding sources: Intramural Targeted Anti-COVID-19 grant, National Institutes of Health
Temesgen E. Andargie, Naoko Tsuji, Fayaz Seifuddin, Moon Kyoo Jang, Peter S.T. Yuen, Hyesik Kong, Ilker Tunc, Komudi Singh, Ananth Charya, Kenneth J. Wilkins, Steven D. Nathan, Andrea L. Cox, Mehdi Pirooznia, Robert A. Star, Sean Agbor-Enoh
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.
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
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.
Samuel T. Pellom, Claire Smalley Rumfield, Y. Maurice Morillon II, Nicholas Roller, Lisa K. Poppe, Douglas E. Brough, Helen Sabzevari, Jeffrey Schlom, Caroline Jochems
Complexity of lung microenvironment and changes in cellular composition during disease make it exceptionally hard to understand molecular mechanisms driving development of chronic lung diseases. Although recent advances in cell-type resolved approaches hold great promise for studying complex diseases, their implementation relies on local access to fresh tissue, as traditional tissue storage methods do not allow viable cell isolation. To overcome these hurdles, we developed a versatile workflow that allows storage of lung tissue with high viability, permits thorough sample quality check before cell isolation, and befits sequencing-based profiling. We demonstrate that cryopreservation enables isolation of multiple cell types from both healthy and diseased lungs. Basal cells from cryopreserved airways retain their differentiation ability, indicating that cellular identity is not altered by cryopreservation. Importantly, using RNA sequencing and EPIC Array, we show that gene expression and DNA methylation signatures are preserved upon cryopreservation, emphasizing the suitability of our workflow for -omics profiling of lung cells. Moreover, we obtained high-quality single-cell RNA sequencing data of cells from cryopreserved human lung, demonstrating that cryopreservation empowers single-cell approaches. Overall, thanks to its simplicity, our workflow is well-suited for prospective tissue collection by academic collaborators and biobanks, opening worldwide access to viable human tissue.
Maria Llamazares Prada, Elisa Espinet, Vedrana Mijošek, Uwe Schwartz, Pavlo Lutsik, Raluca Tamas, Mandy Richter, Annika Behrendt, Stephanie T. Pohl, Naja P. Benz, Thomas Muley, Arne Warth, Claus Peter Heußel, Hauke Winter, Jonathan J. M. Landry, Felix J.F. Herth, Tinne C.J. Mertens, Harry Karmouty-Quintana, Ina Koch, Vladimir Benes, Jan O. Korbel, Sebastian M. Waszak, Andreas Trumpp, David M. Wyatt, Heiko F. Stahl, Christoph Plass, Renata Z. Jurkowska
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.
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
In this issue, Khan et al. report a comprehensive analysis of antibody responses in children and adults to the four endemic human coronaviruses. Antibody repertoires differed between children and adults, with children more frequently having antibodies that target functionally important and structurally conserved regions of the spike, nucleocapsid, and matrix proteins. Moreover, they found antibodies targeting a highly conserved region of the spike protein that were cross-reactive among multiple coronaviruses, suggesting a potential important target for vaccine design. The cover image is an illustration of antibodies and a coronavirus. Image credit: Tatiana Shepeleva/Shutterstock.
JCI This Month is a digest of the research, reviews, and other features published each month.