Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.
Sirle Saul, Marwah Karim, Luca Ghita, Pei-Tzu Huang, Winston Chiu, Verónica Durán, Chieh-Wen Lo, Sathish Kumar, Nishank Bhalla, Pieter Leyssen, Farhang Alem, Niloufar A. Boghdeh, Do HoangNhu Tran, Courtney A. Cohen, Jacquelyn A. Brown, Kathleen E. Huie, Courtney Tindle, Mamdouh Sibai, Chengjin Ye, Ahmed Magdy Khalil, Kevin Chiem, Luis Martinez-Sobrido, John M. Dye, Benjamin A. Pinsky, Pradipta Ghosh, Soumita Das, David E. Solow-Cordero, Jing Jin, John P. Wikswo, Dirk Jochmans, Johan Neyts, Steven De Jonghe, Aarthi Narayanan, Shirit Einav
The volume and composition of a thin layer of liquid covering the airway surface defend the lung from inhaled pathogens and debris. Airway epithelia secrete Cl– into the airway surface liquid through CFTR (cystic fibrosis transmembrane conductance regulator) channels, thereby increasing the volume of airway surface liquid. The discovery that pulmonary ionocytes contain high levels of CFTR led us to predict that ionocytes drive secretion. However, we found the opposite. Elevating ionocyte abundance increased liquid absorption, whereas reducing ionocyte abundance increased secretion. In contrast to other airway epithelial cells, ionocytes contained barttin/Cl– channels in their basolateral membrane. Disrupting barttin/Cl– channel function impaired liquid absorption, and overexpressing barttin/Cl– channels increased absorption. Together, apical CFTR and basolateral barttin/Cl– channels provide an electrically conductive pathway for Cl– flow through ionocytes, and the transepithelial voltage generated by apical Na+ channels drives absorption. These findings indicate that ionocytes mediate liquid absorption, and secretory cells mediate liquid secretion. Segregating these counteracting activities to distinct cell types enables epithelia to precisely control the airway surface. Moreover, the divergent role of CFTR in ionocytes and secretory cells suggests that cystic fibrosis disrupts both liquid secretion and absorption.
Lei Lei, Soumba Traore, Guillermo S. Romano Ibarra, Philip H. Karp, Tayyab Rehman, David K. Meyerholz, Joseph Zabner, David A. Stoltz, Patrick L. Sinn, Michael J. Welsh, Paul B. McCray, Jr., Ian M. Thornell
The nucleus accumbens (NAc) is the most promising target for drug use disorder treatment. Deep brain stimulation (DBS) of NAc is effective for drug use disorder treatment. However, the mechanisms by which DBS produces its therapeutic effects remain enigmatic. Here, we define a behavioral cut-off criterion to distinguish depressive-like behaviors and nondepressive-like behaviors in mice after morphine withdrawal. We identified a basolateral amygdala (BLA) to NAc D1 medium spiny neuron (D1 MSN) pathway that controls depressive-like behaviors after morphine withdrawal. Furthermore, the paraventricular nucleus of thalamus (PVT) to NAc D2 MSN pathway that controls naloxone-induced acute withdrawal symptoms. Optogenetic-induced long-term potentiation with kappa-opioid receptor (KOR) antagonism enhanced BLA to NAc D1 MSN signaling and also altered the excitation/inhibition balance of NAc D2 MSN signaling. We also verified that a new 50 Hz DBS protocol reversed morphine withdrawal-evoked abnormal plasticity in NAc. Importantly, this refined DBS treatment effectively alleviated naloxone-induced withdrawal symptoms and depressive-like behaviors and prevented stress-induced reinstatement. Taken together, the results demonstrated that input- and cell type-specific synaptic plasticity underlies morphine withdrawal, which may lead to novel targets for the treatment of opioid use disorder.
Yongsheng Zhu, Kejia Wang, Tengfei Ma, Yuanyuan Ji, Yin Lou, Xiaoyu Fu, Ye Lu, Yige Liu, Wei Dang, Qian Zhang, Fangyuan Yin, Kena Wang, Bing Yu, Hongbo Zhang, Jianghua Lai, Yunpeng Wang
B cell clonal expansion and cerebrospinal fluid (CSF) oligoclonal IgG bands are established features of the immune response in multiple sclerosis (MS). Clone-specific IgG1 monoclonal recombinant antibodies (rAbs) derived from MS patient CSF plasmablasts bound to conformational proteolipid protein 1 (PLP1) membrane complexes and, when injected into mouse brain with human complement, recapitulated histologic features of MS pathology: oligodendrocyte cell loss, complement deposition, and CD68+ phagocyte infiltration. Conformational PLP1 membrane epitopes were complex and governed by the local cholesterol and glycolipid microenvironment. Antibodies against conformational PLP1 membrane complexes targeted multiple surface epitopes, were enriched within the CSF compartment, and were detected in most MS patients but not in inflammatory and non-inflammatory neurologic controls. CSF PLP1 complex antibodies provide a pathogenic autoantibody biomarker specific for MS.
Gregory P. Owens, Timothy J. Fellin, Adeline Matschulat, Vanessa Salas, Kristin L. Schaller, Katherine S. Given, Alanna M. Ritchie, Andre Navarro, Kevin Blauth, Ethan G. Hughes, Wendy B. Macklin, Jeffrey L. Bennett
Clinical genome editing is emerging for rare disease treatment, but one of the major limitations is the targeting of CRISPR editors delivery. We delivered base editors to the retinal pigmented epithelium (RPE) in the mouse eye using silica nanocapsules (SNC) as a treatment for retinal degeneration. Leber Congenital Amaurosis (LCA16) is a rare pediatric blindness caused by point mutations in the KCNJ13 gene, a loss-of-function inwardly rectifying potassium channel (Kir7.1) in the RPE. SNC carrying adenine base editor (ABE8e) mRNA and single-guide RNA precisely and efficiently corrected KCNJ13W53X/W53X mutation. Editing in both patient fibroblasts (47%) and human-induced pluripotent stem cell-derived RPE (LCA16-iPSC-RPE) (17%) had a negligible off-target response. We detected functional Kir7.1 channels in the edited LCA16-iPSC-RPE. In the LCA16 mouse model (Kcnj13W53X/+∆R), RPE cells targeted SNC delivery of ABE8e mRNA preserved normal visual function measured by full-field electroretinogram (ERG). Moreover, multifocal ERG confirmed the topographic measure of electrical activity primarily originating from the edited retinal area at the injection site. Preserved retina structure, post-treatment, was established by Optical Coherence Tomography (OCT). This preclinical validation of targeted ion channel functional rescue, a challenge for pharmacological and genomic interventions, reinforced the effectiveness of nonviral genome editing therapy for rare inherited disorders.
Meha Kabra, Pawan K. Shahi, Yuyuan Wang, Divya Sinha, Allison Spillane, Gregory A. Newby, Shivani Saxena, Yao Tong, Yu Chang, Amr A Abdeen, Kimberly L. Edwards, Cole O. Theisen, David R. Liu, David M. Gamm, Shaoqin Gong, Krishanu Saha, Bikash R. Pattnaik
Lung inflammation is a hallmark of Coronavirus disease 2019 (COVID-19) in severely ill patients and the pathophysiology of disease is thought to be immune-mediated. Mast cells (MCs) are polyfunctional immune cells present in the airways, where they respond to certain viruses and allergens, often promoting inflammation. We observed widespread degranulation of MCs during acute and unresolved airway inflammation in SARS-CoV-2-infected mice and non-human primates. Using a mouse model of MC-deficiency, MC-dependent interstitial pneumonitis, hemorrhaging, and edema in the lung were observed during SARS-CoV-2 infection. In humans, transcriptional changes in patients requiring oxygen supplementation also implicated cells with a MC phenotype in severe disease. MC activation in humans was confirmed, through detection of MC-specific proteases, including chymase, levels of which were significantly correlated with disease severity and with biomarkers of vascular dysregulation. These results support the involvement of MCs in lung tissue damage during SARS-CoV-2 infection in animal models and the association of MC activation with severe COVID-19 in humans, suggesting potential strategies for intervention.
Janessa Yan Jun Tan, Danielle E. Anderson, Abhay P.S. Rathore, Aled O'Neill, Chinmay Kumar Mantri, Wilfried A.A. Saron, Cheryl Q.E. Lee, Wern Cui Chu, Adrian E.Z. Kang, Randy Foo, Shirin Kalimuddin, Jenny G. Low, Lena Ho, Paul Tambyah, Thomas W. Burke, Christopher W. Woods, Kuan Rong Chan, Joern Karhausen, Ashley L. St. John
Adoptive immunotherapy with Tregs is a promising approach for prevention or treatment of type 1 diabetes. Islet antigen-specific Tregs have more potent therapeutic effects than polyclonal cells, but their low frequency is a barrier for clinical application. To generate Tregs that recognize islet antigens, we engineered a chimeric antigen receptor (CAR) derived from a monoclonal antibody with specificity for the insulin B-chain 10-23 peptide presented in the context of the IAg7 MHC class II allele present in NOD mice. Peptide specificity of the resulting InsB-g7 CAR was confirmed by tetramer staining and T cell proliferation in response to recombinant or islet-derived peptide. The InsB-g7 CAR re-directed NOD Treg specificity such that insulin B 10-23-peptide stimulation enhanced suppressive function, measured via reduction of proliferation and IL-2 production by BDC2.5 T cells and CD80 and CD86 expression on dendritic cells. Co-transfer of InsB-g7 CAR Tregs prevented adoptive transfer diabetes by BDC2.5 T cells in immunodeficient NOD mice. In wild type NOD mice, InsB-g7 CAR Tregs prevented spontaneous diabetes. These results show that engineering Treg specificity for islet antigens using a T cell receptor-like CAR is a promising new therapeutic approach for the prevention of autoimmune diabetes.
Justin A. Spanier, Vivian Fung, Christine M. Wardell, Mohannad H. Alkhatib, Yixin Chen, Linnea A. Swanson, Alexander J. Dwyer, Matthew E. Weno, Nubia Silva, Jason S. Mitchell, Paul C. Orban, Majid Mojibian, C. Bruce Verchere, Brian T. Fife, Megan K. Levings
BACKGROUND. Recurrent and/or metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) is often an incurable disease, with patients experiencing median survival of under ten months and significant morbidity. While immune checkpoint blockade (ICB) drugs are effective in ~20% of patients, the remaining experience limited clinical benefit and are exposed to potential adverse effects and financial costs. Clinically approved biomarkers, such as tumor mutational burden (TMB), have modest predictive value in HNSCC. METHODS. We analyze clinical and genomic features, generated using whole-exome sequencing, in 133 ICB-treated R/M HNSCC patients, of whom 69 had virus-associated and 64 had non-virus-associated tumors. RESULTS. Hierarchical clustering of genomic data revealed six molecular subtypes characterized by a wide range of objective response rates and survival after ICB therapy. The prognostic importance of these 6 subtypes was validated in an external cohort. A random forest-based predictive model, using several clinical and genomic features, predicted progression-free survival (PFS), overall survival (OS), and response with greater accuracy than a model based on TMB alone. Recursive partitioning analysis identified three features (systemic inflammatory response index, TMB, and smoking signature) that classified patients into risk groups with accurate discrimination of PFS and OS. CONCLUSION. These findings shed light on the immunogenomic characteristics of HNSCC tumors that drive differential response to ICB and identify a clinical-genomic classifier that outperforms the current clinically approved biomarker of TMB. This validated predictive tool may help with clinical risk stratification in patients with R/M HNSCC for whom ICB is being considered.
Cristina Valero, Mahdi Golkaram, Joris L. Vos, Bin Xu, Conall Fitzgerald, Mark Lee, Shannon K. Kaplan, Catherine Y Han, Xin Pei, Reith Sarkar, Lillian A. Boe, Abhinav Pandey, Elizabeth S. Koh, Charlotte L. Zuur, David B. Solit, Traci Pawlowski, Li Liu, Alan L. Ho, Diego Chowell, Nadeem Riaz, Timothy A. Chan, Luc G.T. Morris
BACKGROUND. Warts, Hypogammaglobulinemia, Infections and Myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is neutropenia from neutrophil retention in bone marrow and is associated with lymphopenia and monocytopenia. The CXCR4 antagonist plerixafor mobilizes leukocytes to the blood; however, safety and efficacy in WHIM syndrome are undefined. METHODS. In this investigator-initiated, single-center, randomized, quadruple-masked phase 3 crossover trial, we compared the total infection severity score (TISS) as primary endpoint in an intent-to-treat manner in 19 WHIM patients for 12-months on plerixafor versus 12-months on G-CSF, the standard-of-care for severe congenital neutropenia. RESULTS. Plerixafor was non-superior to G-CSF for TISS (p=0.65). In exploratory endpoints, plerixafor was non-inferior to G-CSF for maintaining neutrophil counts >500 cells/microliter (p=0.023) and was superior to G-CSF for maintaining lymphocyte counts >1000 cells/microliter (p<0.0001). Complete regression of a subset of large wart areas occurred on plerixafor in 5 of 7 patients with major wart burdens at baseline. Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life, or the incidence of drug failure or serious adverse events. CONCLUSIONS. Plerixafor was not superior in WHIM patients to G-CSF for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results support continued study of plerixafor as a potential treatment for WHIM syndrome.(Funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases; clinicaltrials.gov registration number, NCT02231879)
David H. McDermott, Daniel Velez, Elena Cho, Edward W. Cowen, John J. DiGiovanna, Diana V. Pastrana, Christopher B. Buck, Katherine R. Calvo, Pamela J. Gardner, Sergio D. Rosenzweig, Pamela Stratton, Melissa A. Merideth, H. Jeffrey Kim, Carmen Brewer, James D. Katz, Douglas B. Kuhns, Harry L. Malech, Dean Follmann, Michael P. Fay, Philip M. Murphy
Cystinosis is a lysosomal storage disease that is characterized by the accumulation of di-peptide cystine within the lumen. It is caused by mutations in the cystine exporter, cystinosin. Most of the clinically reported mutations are due to the loss of transporter function. In this study, we identified a rapidly degrading disease variant, referred to as cystinosin(7∆). We demonstrated that this mutant is retained in the endoplasmic reticulum (ER) and degraded via the ER-associated degradation (ERAD) pathway. Using genetic and chemical inhibition methods, we elucidated the role of HRD1, p97, EDEMs, and the proteasome complex in cystinosin(7∆) degradation pathway. Having understood the degradation mechanisms, we tested some chemical chaperones, previously used for treating CFTR F508∆, and demonstrated that they could facilitate the folding and trafficking of cystinosin(7∆). Strikingly, chemical chaperone treatment can reduce the lumenal cystine level by ~70%. Conclusively, our study establishes the connection between ERAD and cystinosis pathogenesis and demonstrates the possibility of using chemical chaperones to treat cystinosin(7∆).
Varsha Venkatarangan, Weichao Zhang, Xi Yang, Jess G. Thoene, Si H. Hahn, Ming Li
Negative regulation of exocytosis from secretory cells is accomplished through inhibitory signals from Gi/o G-protein-coupled-receptors by Gβγ subunit inhibition of two mechanisms: decreased calcium entry and direct interaction of Gβγ with Soluble N-ethylmaleimide-sensitive factor Attachment Protein (SNAP) Receptor (SNARE) plasma membrane fusion machinery. Previously, we disabled the second mechanism with a SNAP25 truncation (SNAP25Δ3) decreasing Gβγ affinity for the SNARE complex, leaving exocytotic fusion and modulation of calcium entry intact, and removing GPCR-Gβγ inhibition of SNARE-mediated exocytosis. Here, we report substantial metabolic benefit in mice carrying this mutation. SNAP25Δ3/Δ3 mice exhibit enhanced insulin sensitivity and beiging of white fat. Metabolic protection was amplified in SNAP25Δ3/Δ3 mice challenged with high fat diet. Glucose homeostasis, whole body insulin action, and insulin-mediated glucose uptake into white adipose tissue were improved along with resistance to diet-induced obesity. Metabolic protection in SNAP25Δ3/Δ3 mice occurred without compromising the physiological response to fasting or cold. All metabolic phenotypes were reversed at thermoneutrality, suggesting basal autonomic activity is required. Direct electrode stimulation of sympathetic neuron exocytosis from SNAP25Δ3/Δ3 inguinal adipose depots resulted in enhanced and prolonged norepinephrine release. Thus, the Gβγ-SNARE interaction represents a cellular mechanism that deserves further exploration as an additional avenue for combatting metabolic disease.
Ryan P. Ceddia, Zack Zurawski, Analisa Thompson Gray, Feyisayo Adegboye, Ainsley McDonald-Boyer, Fubiao Shi, Dianxin Liu, Jose Maldonado, Jiesi Feng, Yulong Li, Simon Alford, Julio E. Ayala, Owen P. McGuinness, Sheila Collins, Heidi E. Hamm
Biological aging can be described as accumulative, prolonged metabolic stress, and is the major risk factor for cognitive decline and Alzheimer’s disease (AD). Recently, we identified and described a quinone reductase 2 (QR2) pathway in the brain, in which QR2 acts as a removable memory constraint and metabolic buffer within neurons. QR2 becomes over-expressed with age, and is possibly a novel contributing factor to age-related metabolic stress and cognitive deficit. We found that in human cells, genetic removal of QR2 produces a shift in the proteome opposing that found in AD brains, while simultaneously reducing oxidative stress. We therefore created highly specific QR2 inhibitors (QR2i’s), enabling evaluation of chronic QR2 inhibition as a novel way to reduce biological-age related metabolic stress and cognitive decline. QR2i’s replicated results obtained by genetic removal of QR2 while local QR2i microinjection improved hippocampal and cortical dependent learning in rats and mice. Continuous consumption of QR2i’s in drinking-water improved cognition and reduced pathology in the brains of AD-model mice (5xFAD), with a noticeable between-sex effect on treatment duration. These results demonstrate the importance of QR2 activity- and pathway function in the healthy and neurodegenerative brain, and the great therapeutic potential of QR2i’s as first-in-class drugs.
Nathaniel L. Gould, Gila R. Scherer, Silvia Carvalho, Khriesto Shurrush, Haneen Kayyal, Efrat Edry, Alina Elkobi, Orit David, Maria Foqara, Darshit Thakar, Tommaso Pavesi, Vijendra Sharma, Matthew Walker, Matthew Maitland, Orly Dym, Shira Albeck, Yoav Peleg, Nicolas Germain, Ilana Babaev, Haleli Sharir, Maya Lalzar, Boris Shklyar, Neta Hazut, Mohammad Khamaisy, Maxime Lévesque, Gilles Lajoie, Massimo Avoli, Gabriel Amitai, Bruce Lefker, Chakrapani Subramanyam, Brian Shilton, Haim Barr, Kobi Rosenblum
Glial activation and inflammation coincide with neurofibrillary tangles (NFT) formation in neurons. However, the mechanism behind tau fibril and glia interaction is poorly understood. Here, we found that tau preformed fibrils (PFF) caused induction of inflammation in microglia by specifically activating the TLR2-MyD88, but not TLR4-MyD88, pathway. Accordingly, TLR2 interacting domain of MyD88 (wtTIDM) peptide inhibited tau PFF-induced activation of TLR2-MyD88-NF-κB pathway resulting in reduced inflammation. Nasal administration of wtTIDM in P301S tau-expressing PS19 mice was found to inhibit gliosis and inflammatory markers, along with reduction of pathogenic tau in the hippocampus, resulting in improved cognitive behavior in PS19 mice. The inhibitory effect of wtTIDM on tau pathology was absent in PS19 mice lacking TLR2, reinforcing the essential involvement of TLR2 in wtTIDM- mediated effects in vivo. While understanding the mechanism further, we found that tau promoter harboured a potential NF-κB binding site and that proinflammatory molecules increased the transcription of tau in neurons via NF-κB. These results suggest that tau-induced neuroinflammation and neuropathology require TLR2 and that neuroinflammation directly upregulates tau in neurons via NF-κB, highlighting a direct connection between inflammation and tauopathy.
Debashis Dutta, Malabendu Jana, Ramesh Kumar Paidi, Moumita Majumder, Sumita Raha, Sridevi Dasarathy, Kalipada Pahan
Clonal hematopoiesis of indeterminate potential (CHIP) is associated with an increased risk of cardiovascular diseases (CVD), putatively via inflammasome activation. We pursued an inflammatory gene modifier scan for CHIP-associated CVD risk among 424,651 UK Biobank participants. CHIP was identified using whole exome sequencing data of blood DNA and modeled both as a composite and for common drivers (DNMT3A, TET2, ASXL1, and JAK2) separately. We developed predicted gene expression scores for 26 inflammasome-related genes and assessed how they modify CHIP-associated CVD risk. We identify IL1RAP as a potential key molecule for CHIP-associated CVD risk across genes and increased AIM2 gene expression leading to heightened JAK2- and ASXL1-associated CVD risks. We show that CRISPR-induced Asxl1 mutated murine macrophages have a particularly heightened inflammatory response to AIM2 agonism, associated with an increased DNA damage response, as well as increased IL-10 secretion, mirroring a CVD protective effect of IL10 expression in ASXL1 CHIP. Our study supports the role of inflammasomes in CHIP-associated CVD and provides new evidence to support gene-specific strategies to address CHIP-associated CVD risk.
Zhi Yu, Trevor P. Filder, Yunfeng Ruan, Caitlyn Vlasschaert, Tetsushi Nakao, Md Mesbah Uddin, Taralynn Mack, Abhishek Niroula, J. Brett Heimlich, Seyedeh M. Zekavat, Christopher J. Gibson, Gabriel K. Griffin, Yuxuan Wang, Gina M. Peloso, Nancy Heard-Costa, Daniel Levy, Ramachandran S. Vasan, François Aguet, Kristin G. Ardlie, Kent D. Taylor, Stephen S. Rich, Jerome I. Rotter, Peter Libby, Siddhartha Jaiswal, Benjamin L. Ebert, Alexander G. Bick, Alan R. Tall, Pradeep Natarajan
The tumor extracellular matrix (ECM) critically regulates cancer progression and treatment response. Expression of the basement membrane component collagen XVIII (ColXVIII) is induced in solid tumors, but its involvement in tumorigenesis has remained elusive. We show here that ColXVIII is markedly upregulated in human breast cancer (BC) and is closely associated with a poor prognosis in high-grade BCs. We discovered a role for ColXVIII as a modulator of EGFR/ErbB receptor tyrosine kinase signaling and show that it forms a complex with EGFR, HER2 and α6 integrin to promote cancer cell proliferation in a pathway involving its N-terminal portion and the MAPK/ERK1/2 and PI3K/AKT cascades. Studies with Col18a1 mouse models crossed with the MMTV-PyMT mammary carcinogenesis model showed that ColXVIII promotes BC growth and metastasis in a tumor cell-autonomous manner. Moreover, the number of mammary cancer stem cells was significantly reduced in the MMTV-PyMT and human cell models upon ColXVIII inhibition. Finally, ablation of ColXVIII substantially improved the efficacy of ErbB-targeting therapies in both preclinical models. In summary, ColXVIII was found to sustain the stemness properties of BC cells, and tumor progression and metastasis through ErbB signaling, suggesting that targeting ColXVIII in the tumor milieu may have important therapeutic potential.
Raman Devarajan, Valerio Izzi, Hellevi Peltoketo, Gunilla Rask, Saila Kauppila, Marja-Riitta Väisänen, Heli Ruotsalainen, Guillermo A. Martinez-Nieto, Sanna-Maria Karppinen, Timo Väisänen, Inderjeet Kaur, Jussi Koivunen, Takako Sasaki, Robert Winqvist, Aki Manninen, Fredrik Wärnberg, Malin Sund, Taina Pihlajaniemi, Ritva Heljasvaara
X-linked myotubular myopathy (XLMTM) is a fatal congenital disorder caused by mutations in the MTM1 gene. Currently, there are no approved treatments, though AAV8-mediated gene transfer therapy has shown promise in animal models and preliminarily in patients. However, four patients with XLMTM treated with gene therapy have died from progressive liver failure, and hepatobiliary disease has now been recognized more broadly in association with XLMTM. In an attempt to understand whether loss of MTM1 itself is associated with liver pathology, we have characterized a novel liver phenotype in a zebrafish model of this disease. Specifically, we have found that loss-of-function mutations in mtm1 lead to severe liver abnormalities including impaired bile flux, structural abnormalities of the bile canaliculus, and improper endosomal-mediated trafficking of canalicular transporters. Using a reporter tagged Mtm1 zebrafish line, we have established localization of Mtm1 in the liver in association with Rab11 and canalicular transport proteins, and demonstrated that hepatocyte specific re-expression of Mtm1 can rescue the cholestatic phenotype. Lastly, we completed a targeted chemical screen, and found that Dynasore, a dynamin II inhibitor, is able to partially restore bile flow and transporter localization to the canalicular membrane. In summary, we demonstrate for the first time liver abnormalities that are directly caused by MTM1 mutation in a pre-clinical model, thus establishing the critical framework for better understanding and comprehensive treatment of the human disease.
Sophie Karolczak, Ashish R. Deshwar, Evangelina Aristegui, Binita M. Kamath, Michael W. Lawlor, Gaia Andreoletti, Jonathan R. Volpatti, Jillian L. Ellis, Chunyue Yin, James J. Dowling
Vascular networks form, remodel and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with Bone Morphogenic Protein 9 (BMP9) and BMP10. Conversely, mutation of the BMP receptors ALK1, Endoglin or the downstream effector SMAD4 leads to Hereditary Hemorrhagic Telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). But how endothelial cells (EC) integrate FSS and BMP signals in vascular development and homeostasis, and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between fluid shear stress and SMAD signaling in vascular stability and its failure in HHT. We have now found that loss of Smad4 increases ECs’ sensitivity to flow by lowering the FSS set point with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling leading to cell cycle progression - mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors, CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.
Kuheli Banerjee, Yanzhu Lin, Johannes Gahn, Julio Cordero, Purnima Gupta, Islam Mohamed, Mariona Graupera, Gergana Dobreva, Martin A. Schwartz, Roxana Ola
Sickle cell disease (SCD) is a hereditary hemoglobinopathy characterized by painful vaso-occlusive crises (VOC) and chronic hemolysis. The mononuclear phagocyte system is pivotal to SCD pathophysiology, but the mechanisms governing monocyte/macrophage differentiation remain unknown. This study examined the influence of hemolysis on circulating monocyte trajectories in SCD. We discovered that hemolysis stimulated CSF-1 production, partly by endothelial cells via Nrf2, promoting classical monocyte (CMo) differentiation into blood patrolling monocytes (PMo) in SCD mice. However, hemolysis also upregulated CCL-2 through IFN-I, inducing CMo transmigration and differentiation into tissue monocyte-derived macrophages. Blocking CMo transmigration by anti-P-selectin antibody in SCD mice increased circulating PMo, corroborating that CMo-to-tissue macrophage differentiation occurs at the expense of CMo-to-blood PMo differentiation. We observed a positive correlation between plasma CSF-1/CCL-2 ratios and blood PMo levels in SCD patients, underscoring the clinical significance of these two opposing factors in monocyte differentiation. Combined treatment with CSF-1 and anti-P-selectin antibody more effectively increased PMo numbers and reduced stasis compared to single-agent therapies in SCD mice. Altogether, these data indicate that monocyte fates are regulated by the balance between two heme pathways, Nrf2-CSF-1 and IFN-I-CCL-2, and suggest that the CSF-1/CCL-2 ratio may present a diagnostic and therapeutic target in SCD.
Yunfeng Liu, Shan Su, Sarah Shayo, Weili Bao, Mouli Pal, Kai Dou, Patricia A. Shi, Banu Aygun, Sally Campbell-Lee, Cheryl A. Lobo, Avital Mendelson, Xiuli An, Deepa Manwani, Hui Zhong, Karina Yazdanbakhsh
Glucocorticoids are steroid hormones with potent immunosuppressive properties. Their primary source is the adrenals, where they are generated via de novo synthesis from cholesterol. In addition, many tissues have a recycling pathway in which glucocorticoids are regenerated from inactive metabolites by the enzyme 11β-HSD1 (encoded by Hsd11b1). Here we find that multiple tumor types express Hsd11b1 and produce active glucocorticoids. Genetic ablation of Hsd11b1 in such cells had no effect on in vitro growth but reduced in vivo tumor progression, which corresponded with increased frequencies of tumor-infiltrating CD8+ T cells (TIL) expressing activation markers and producing effector cytokines. Tumor-derived glucocorticoids were found to promote signatures of Treg activation and suppress signatures of Tconv activation in tumor-infiltrating Treg. Indeed, CD8+ T cell activation was restored and tumor growth reduced in mice with Treg-specific glucocorticoid receptor deficiency. Importantly, pharmacologic inhibition of 11β-HSD1 reduced tumor growth to the same degree as gene knockout, and rendered immunotherapy-resistant tumors susceptible to PD-1 blockade. Given that HSD11B1 expression is upregulated in many human tumors and that inhibition of 11β-HSD1 is well-tolerated in clinical studies, these data suggest that targeting 11β-HSD1 may be a beneficial adjunct in cancer therapy.
Matthew D. Taves, Shizuka Otsuka, Michaela A. Taylor, Kaitlynn M. Donahue, Thomas J. Meyer, Margaret C. Cam, Jonathan D. Ashwell
Protease activated receptor (PAR) 4 (gene: F2RL3) harbors a functional dimorphism, rs773902 A/G (encoding Thr120/Ala120, respectively) and is associated with greater platelet aggregation. The A allele frequency is more common in Black individuals, and Black individuals have a higher incidence of ischemic stroke than White individuals. However, it is not recognized whether the A allele is responsible for worse stroke outcomes. To directly test the in vivo effect of this variant on stroke, we generated mice where F2rl3 was replaced by F2RL3, thereby expressing human PAR4 (hPAR4) with either Thr120 or Ala120. Compared to hPAR4 Ala120 mice, hPAR4 Thr120 mice had worse stroke outcomes, mediated in part by enhanced platelet activation and platelet-neutrophil interactions. Analyses of 7620 Black subjects with 487 incident ischemic strokes demonstrated the AA genotype was a risk for incident ischemic stroke and worse functional outcomes. In humanized mice, ticagrelor with or without aspirin improved stroke outcomes in hPAR4 Ala120 mice, but not in hPAR4 Thr120 mice. P-selectin blockade improved stroke outcomes and reduced platelet-neutrophil interactions in hPAR4 Thr120 mice. Our results may explain some of the racial disparity in stroke and support the need for studies of non-standard anti-platelet therapies for patients expressing PAR4 Thr120.
Frederik Denorme, Nicole D. Armstrong, Michelle L. Stoller, Irina Portier, Emilia A. Tugolukova, Rikki M. Tanner, Emilie Montenont, Seema Bhatlekar, Mark Cody, John L. Rustad, Abigail Ajanel, Neal D. Tolley, Darian C. Murray, Julie L. Boyle, Marvin T. Nieman, Steven E. McKenzie, Christian Con Yost, Leslie A. Lange, Mary Cushman, Marguerite R. Irvin, Paul F. Bray, Robert A. Campbell