Emerging data indicate that complement and neutrophils contribute to the maladaptive immune response that fuels hyper-inflammation and thrombotic microangiopathy, thereby increasing COVID-19 mortality. Here, we investigated how complement interacts with the platelet/neutrophil extracellular traps (NETs)/thrombin axis, using COVID-19 specimens, cell-based inhibition studies and NETs/human aortic endothelial cell (HAEC) co-cultures. Increased plasma levels of NETs, tissue factor (TF) activity and sC5b-9 were detected in patients. Neutrophils of patients yielded high TF expression and released NETs carrying active TF. Treatment of control neutrophils with COVID-19 platelet-rich plasma generated TF-bearing NETs that induced thrombotic activity of HAEC. Thrombin or NETosis inhibition or C5aR1 blockade attenuated platelet-mediated NET-driven thrombogenicity. COVID-19 serum induced complement activation in vitro, consistent with high complement activity in clinical samples. Complement C3 inhibition with compstatin Cp40 disrupted TF expression in neutrophils. In conclusion, we provide a mechanistic basis for a pivotal role of complement and NETs in COVID-19 immunothrombosis. This study supports strategies against SARS-CoV-2 that exploit complement or NETosis inhibition.
Panagiotis Skendros, Alexandros Mitsios, Akrivi Chrysanthopoulou, Dimitrios C. Mastellos, Simeon Metallidis, Petros Rafailidis, Maria Ntinopoulou, Eleni Sertaridou, Victoria Tsironidou, Christina Tsigalou, Maria G. Tektonidou, Theocharis Konstantinidis, Charalampos Papagoras, Ioannis Mitroulis, Georgios Germanidis, John D. Lambris, Konstantinos Ritis
Desmoplasia describes the deposition of extensive extracellular matrix and defines primary pancreatic ductal adenocarcinoma (PDA). The acellular component of this stroma has been implicated in PDA pathogenesis and is being targeted therapeutically in clinical trials. By analyzing the stromal content of PDA samples from numerous annotated PDA data sets and correlating stromal content with both anatomic site and clinical outcome, we found PDA metastases in the liver, the primary cause of mortality to have less stroma, have higher tumor cellularity than primary tumors. Experimentally manipulating stromal matrix with an anti– lysyl oxidase like-2 (anti-LOXL2) antibody in syngeneic orthotopic PDA mouse models significantly decreased matrix content, led to lower tissue stiffness, lower contrast retention on computed tomography, and accelerated tumor growth, resulting in diminished overall survival. These studies suggest an important protective role of stroma in PDA and urge caution in clinically deploying stromal depletion strategies.
Honglin Jiang, Robert J. Torphy, Katja Steiger, Henry Hongo, Alexa J. Ritchie, Mark Kriegsmann, David Horst, Sarah E. Umetsu, Nancy M. Joseph, Kimberly McGregor, Michael J. Pishvaian, Edik M. Blais, Brian Lu, Mingyu Li, Michael Hollingsworth, Connor Stashko, Keith Volmar, Jen Jen Yeh, Valerie M. Weaver, Zhen J. Wang, Margaret A. Tempero, Wilko Weichert, Eric A. Collisson
Although broadly protective, stem-targeted Abs against the influenza A virus hemagglutinin (HA) have been well studied, very limited information is available on Abs that broadly recognize the head domain. We determined the crystal structure of the HA protein of the avian H7N9 influenza virus in complex with a pan-H7, non-neutralizing, protective human Ab. The structure revealed a B cell epitope in the HA head domain trimer interface (TI). This discovery of a second major protective TI epitope supports a model in which uncleaved HA trimers exist on the surface of infected cells in a highly dynamic state that exposes hidden HA head domain features.
Jinhui Dong, Iuliia Gilchuk, Sheng Li, Ryan Irving, Matthew T. Goff, Hannah L. Turner, Andrew B. Ward, Robert H. Carnahan, James E. Crowe Jr.
Peripheral neurotoxicity is a debilitating toxicity that afflicts up to 90% of patients with colorectal cancer receiving oxaliplatin-containing therapy. Although emerging evidence has highlighted the importance of various solute carriers to the toxicity of anticancer drugs, the contribution of these proteins to oxaliplatin-induced peripheral neurotoxicity remains controversial. Among candidate transporters investigated in genetically-engineered mouse models, we provide evidence for a critical role of the organic cation transporter 2 (OCT2) in satellite glial cells to oxaliplatin-induced neurotoxicity, and demonstrate that targeting OCT2 using genetic and pharmacological approaches ameliorates acute and chronic forms of neurotoxicity. The relevance of this transport system was verified in transporter-deficient rats as a secondary model organism, and translational significance of preventative strategies was demonstrated in preclinical models of colorectal cancer. These studies suggest that pharmacological targeting of OCT2 could be exploited to afford neuroprotection in cancer patients requiring treatment with oxaliplatin.
Kevin M. Huang, Alix F. Leblanc, Muhammad Erfan Uddin, Ji Young Kim, Mingqing Chen, Eric D. Eisenmann, Alice Gibson, Yang Li, Kristen W. Hong, Duncan DiGiacomo, Sherry Huinan Xia, Paola Alberti, Alessia Chiorazzi, Stephen N. Housley, Timothy C. Cope, Jason A. Sprowl, Jing Wang, Charles L. Loprinzi, Anne Noonan, Maryam Lustberg, Guido Cavaletti, Navjotsingh Pabla, Shuiying Hu, Alex Sparreboom
The microbiome provides resistance to infection. However, mechanisms for this are poorly understood. Here we demonstrate in a murine model that colonization with the intestinal bacterium Clostridium scindens provided protection from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte-progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone-marrow from C. scindens colonized-mice into naïve-mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Because of the known ability of the Lachnoclostridia C. scindens to metabolize the bile salt cholate, we measured deoxycholate and discovered that it was increased in the sera of C. scindens colonized specific pathogen free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone (in the absence of C. scindens) increased GMPs and provided protection from amebiasis. We have discovered a mechanism by which C. scindens and the microbially-metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with Entamoeba histolytica.
Stacey L. Burgess, Jhansi L. Leslie, Md. Jashim Uddin, David Noah Oakland, Carol A. Gilchrist, G. Brett Moreau, Koji Watanabe, Mahmoud M. Saleh, Morgan Simpson, Brandon A. Thompson, David T. Auble, Stephen D. Turner, Natasa Giallourou, Jonathan Swann, Zhen Pu, Jennie Z. Ma, Rashidul Haque, William A. Petri, Jr.
Molecular mechanisms governing the development of mammalian cochlea, the hearing organ, remain largely unknown. Through genome sequencing in three subjects from two families with non-syndromic cochlear aplasia, we identified homozygous 221 KB and 338 KB deletions in a non-coding region on chromosome 8 with an ~200 KB overlapping section. Genomic location of the overlapping deleted region was starting from ~350 KB downstream of GDF6. Otic lineage cells differentiated from induced pluripotent stem cells derived from an affected individual show reduced expression of GDF6 compared to control cells. A mouse knock-out of Gdf6 reveals cochlear aplasia closely resembling the human phenotype. We conclude that GDF6 plays a necessary role in early cochlear development controlled by cis-regulatory elements located within ~500 KB region of the genome in humans and that its disruption leads to deafness due to cochlear aplasia.
Guney Bademci, Clemer Abad, Filiz Basak Cengiz, Serhat Seyhan, Armagan Incesulu, Shengru Guo, Suat Fitoz, Emine Ikbal Atli, Nicholas C. Gosstola, Selma Demir, Brett M. Colbert, Gozde Cosar Seyhan, Claire J. Sineni, Duygu Duman, Hakan Gurkan, Cynthia Casson Morton, Derek M. Dykxhoorn, Katherina Walz, Mustafa Tekin
Infusion of the broadly neutralizing antibody VRC01 has been evaluated in HIV-1 chronically infected individuals. Here we studied how VRC01 infusions impacted viral rebound after cessation of antiretroviral therapy (ART) in 18 acutely-treated and durably-suppressed individuals. Viral rebound occurred in all individuals, yet VRC01 infusions modestly delayed rebound and participants who showed a faster decay of VRC01 in serum rebounded more rapidly (Rho=0.60, p=0.03). Participants with strains most sensitive to VRC01 or with VRC01 epitope motifs similar to known VRC01-susceptible strains rebounded later (Rho=-0.70, p<0.03). Upon rebound, HIV-1 sequences were indistinguishable from those sampled at diagnosis. Across the cohort, participant derived Env showed different sensitivity to VRC01 neutralization (including two resistant viruses), yet neutralization sensitivity was similar at diagnosis and post-rebound, indicating the lack of selection for VRC01-resistance during treatment interruption.Our results showed that viremia rebounded despite the absence of HIV-1 adaptation to VRC01 and an average VRC01 trough of 221µg/mL. While VRC01 levels were insufficient to prevent a resurgent infection, knowledge that they did not mediate Env mutations in acute-like viruses is relevant for antibody-based strategies in acute infection.
Evan M. Cale, Hongjun Bai, Meera Bose, Michael A. Messina, Donn Colby, Eric Sanders-Buell, Bethany L. Dearlove, Yifan Li, Emily Engeman, Daniel Silas, Anne Marie O’Sullivan, Brendan Mann, Suteeraporn Pinyakorn, Jintana Intasan, Khunthalee Benjapornpong, Carlo Sacdalan, Eugene Kroon, Nittaya Phanuphak, Robert Gramzinski, Sandhya Vasan, Merlin L. Robb, Nelson L. Michael, Rebecca M. Lynch, Robert Bailer, Amélie Pagliuzza, Nicolas Chomont, Amarendra Pegu, Nicole A. Doria-Rose, Lydie Trautmann, Trevor A. Crowell, John Mascola, Jintanat Ananworanich, Sodsai Tovanabutra, Morgane Rolland
Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation via cleavage of protease-activated receptors (PAR)1 and PAR2. KLK6 levels are elevated in multiple inflammatory and autoimmune conditions, but no definitive role in pathogenesis has been established. Here, we show that skin-targeted overexpression of KLK6 causes generalized, severe psoriasiform dermatitis with spontaneous development of debilitating psoriatic arthritis-like joint disease. The psoriatic skin and joint phenotypes are reversed by normalization of skin KLK6 levels and attenuated following genetic elimination of PAR1 but not PAR2. Conservation of this regulatory pathway was confirmed in human psoriasis using vorapaxar, an FDA-approved PAR1 antagonist, on explanted lesional skin from psoriasis patients. Beyond defining a critical role for KLK6-PAR1 signaling in promoting psoriasis, our results demonstrate that KLK6-PAR1-mediated inflammation in the skin alone is sufficient to drive inflammatory joint disease. Further, we identify PAR1 as a promising cytokine-independent target in therapy of psoriasis and psoriatic arthritis.
Allison C. Billi, Jessica E. Ludwig, Yi Fritz, Richard Rozic, William R. Swindell, Lam C. Tsoi, Dennis Gruszka, Shahla Abdollahi-Roodsaz, Xianying Xing, Doina Diaconu, Ranjitha Uppala, Maya I. Camhi, Philip A. Klenotic, Mrinal K. Sarkar, M. Elaine Husni, Jose U. Scher, Christine McDonald, J. Michelle Kahlenberg, Ronald J. Midura, Johann E. Gudjonsson, Nicole L. Ward
Tyrosine kinase domain (TKD) mutations contribute to acquired resistance to FMS-like tyrosine kinase 3 (FLT3) inhibitors used to treat FLT3-mutant acute myeloid leukemia (AML). We report a cocrystal structure of FLT3 with a type I inhibitor, NCGC1481, that retained potent binding and activity against FLT3 TKD and gatekeeper mutations. Relative to the current generation of advanced FLT3 inhibitors, NCGC1481 exhibited superior antileukemic activity against the common, clinically relevant FLT3-mutant AML cells in vitro and in vivo.
LaQuita M. Jones, Katelyn Melgar, Lyndsey Bolanos, Kathleen Hueneman, Morgan M. Walker, Jian-Kang Jiang, Kelli M. Wilson, Xiaohu Zhang, Jian Shen, Fan Jiang, Patrick Sutter, Amy Wang, Xin Xu, Gregory J. Tawa, Scott B. Hoyt, Mark Wunderlich, Eric O’Brien, John P. Perentesis, Daniel T. Starczynowski, Craig J. Thomas
Peptide MHC class II–based (pMHCII-based) nanomedicines trigger the formation of multicellular regulatory networks by reprogramming autoantigen-experienced CD4+ T cells into autoimmune disease–suppressing T regulatory type 1 (TR1) cells. We have shown that pMHCII-based nanomedicines displaying liver autoimmune disease–relevant yet ubiquitously expressed antigens can blunt various liver autoimmune disorders in a non–disease-specific manner without suppressing local or systemic immunity against infectious agents or cancer. Here, we show that such ubiquitous autoantigen-specific T cells are also awakened by extrahepatic tissue damage and that the corresponding TR1 progeny can suppress experimental autoimmune encephalomyelitis (EAE) and pancreatic β cell autoreactivity. In mice having EAE, nanomedicines displaying either ubiquitous or CNS-specific epitopes triggered the formation and expansion of cognate TR1 cells and their recruitment to the CNS-draining lymph nodes, sparing their liver-draining counterparts. Surprisingly, in mice having both liver autoimmunity and EAE, liver inflammation sequestered these ubiquitous or even CNS-specific TR1 cells away from the CNS, abrogating their antiencephalitogenic activity. In these mice, only the ubiquitous antigen-specific TR1 cells suppressed liver autoimmunity. Thus, the scope of antigen spreading in autoimmune disorders is larger than previously anticipated, involving specificities expected to be silenced by mechanisms of tolerance; the regulatory activity, but not the retention of autoreactive TR1 cells, requires local autoantigen expression.
Channakeshava Sokke Umeshappa, Jacques Mbongue, Santiswarup Singha, Saswat Mohapatra, Jun Yamanouchi, Justin A. Lee, Roopa Hebbandi Nanjundappa, Kun Shao, Urs Christen, Yang Yang, Kristofor K. Ellestad, Pere Santamaria
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