Long COVID, a type of Post-Acute Sequelae of SARS-CoV-2 (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the mechanisms that drive this inflammation remain unknown. Inflammation during acute Coronavirus Disease 2019 could be exacerbated by microbial translocation (from gut and/or lung) to blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We did not observe a significant elevation in plasma markers of bacterial translocation during PASC. However, we observed higher levels of fungal translocation – measured as β-glucan, a fungal cell wall polysaccharide – in the plasma of individuals experiencing PASC compared to those without PASC or SARS-CoV-2 negative controls. The higher β-glucan correlated with higher inflammation and elevated levels of host metabolites involved in activating N-Methyl-D-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neuro-toxic properties. Mechanistically, β-glucan can directly induce inflammation by binding to myeloid cells (via Dectin-1) and activating Syk/NF-κB signaling. Using a Dectin-1/NF-κB reporter model, we found that plasma from individuals experiencing PASC induced higher NF-κB signaling compared to plasma from negative controls. This higher NF-κB signaling was abrogated by Piceatannol (Syk inhibitor). These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.
Leila B. Giron, Michael J. Peluso, Jianyi Ding, Grace Kenny, Netanel F. Zilberstein, Jane Koshy, Kai Ying Hong, Heather Rasmussen, Gregory E. Miller, Faraz Bishehsari, Robert A. Balk, James N. Moy, Rebecca Hoh, Scott Lu, Aaron R. Goldman, Hsin-Yao Tang, Brandon C. Yee, Ahmed Chenna, John W. Winslow, Christos J. Petropoulos, J. Daniel Kelly, Haimanot Wasse, Jeffrey N. Martin, Qin Liu, Ali Keshavarzian, Alan Landay, Steven G. Deeks, Timothy J. Henrich, Mohamed Abdel-Mohsen
Natural killer (NK) cell exhaustion is caused by chronic exposure to activating stimuli during viral infection, tumorigenesis, and prolonged cytokine treatment. Evidence suggests that exhaustion may play a role in disease progression, however relative to T cell exhaustion, the mechanisms underlying NK cell exhaustion and methods of reversing it are poorly understood. Here, we describe a novel in vitro model of exhaustion that employs plate-bound agonists of the NK cell activating receptors NKp46 and NKG2D to induce canonical exhaustion phenotypes. In this model, prolonged activation results in downregulation of activating receptors, upregulation of checkpoint markers, decreased cytokine production and cytotoxicity in vitro, defects in glycolytic metabolism, and decreased persistence, function, and tumor control in vivo. Furthermore, we discover a beneficial effect of NK cell inhibitory receptor signaling during exhaustion. By simultaneously engaging the inhibitory receptor NKG2A during activation in our model, cytokine production and cytotoxicity defects can be mitigated, suggesting that balancing positive and negative signals integrated by effector NK cells can be beneficial for anti-tumor immunity. Together, these data uncover some of the mechanisms underlying NK cell exhaustion in humans and establish our novel in vitro model as a valuable tool for studying the processes regulating exhaustion.
Jacob A. Myers, Dawn Schirm, Laura Bendzick, Rachel Hopps, Carly Selleck, Peter Hinderlie, Martin Felices, Jeffrey S. Miller
Acute kidney injury increases morbidity and mortality and previous studies have shown that remote ischemic preconditioning (RIPC) reduces the risk of acute kidney injury after cardiac surgery. RIPC increases urinary HMGB1 (high mobility group box protein-1) levels in patients which correlates with kidney protection. Here, we show that RIPC reduces renal ischemia-reperfusion injury and improves kidney function in mice. Mechanistically, RIPC increases HMGB1 levels in the plasma and urine and HMGB1 binds to Toll-like receptor 4 (TLR4) on renal tubular epithelial cells, inducing transcriptomic modulation of renal tubular epithelial cells and providing renal protection, whereas TLR4 activation on non-renal cells was shown to contribute to renal injury. This protection is mediated by activation of induction of AMPK⍺ and NF-kB, which induces the upregulation of Sema5b that triggers a transient, protective G1 cell-cycle arrest. In cardiac surgery patients at high risk for postoperative acute kidney injury, increased HMGB1 and Sema5b levels after RIPC were associated with renal protection after surgery. The results may help to develop future clinical treatment options for acute kidney injury.
Jan Rossaint, Melanie Meersch, Katharina Thomas, Sina Mersmann, Martin Lehmann, Jennifer Skupski, Tobias Tekath, Peter Rosenberger, John A. Kellum, Hermann Pavenstädt, Alexander Zarbock
Although macrophages are undoubtedly attractive therapeutic targets for acute kidney injury (AKI) because of their critical roles in renal inflammation and repair, the underlying mechanisms of macrophage phenotype switching and efferocytosis in the regulation of inflammatory responses during AKI are still largely unclear. The present study was to elucidate the role of JAML (junctional adhesion molecule-like protein) in the pathogenesis of AKI. We found that JAML was significantly up-regulated in the kidney from two different murine AKI models including renal ischemia/reperfusion injury (IRI) and cisplatin-induced AKI. By generation of bone marrow chimeric mice, macrophage-specific and tubular-specific Jaml conditional knockout mice, we demonstrated JAML promotes AKI mainly via a macrophage-dependent mechanism and found that JAML-mediated macrophage phenotype polarization and efferocytosis is one of critical signal transduction pathways linking inflammatory responses to AKI. Mechanistically, the effects of JAML on the regulation of macrophages was at least in part, associated with a Mincle-dependent mechanism. Collectively, our studies for the first time explore new biological functions of JAML in macrophages and conclude that JAML is an important mediator and biomarker of AKI. Pharmacologic targeting of JAML mediated signaling pathways at multiple levels may provide a novel therapeutic strategy for patients with AKI.
Wei Huang, Bi-Ou Wang, Yunfeng Hou, Yi Fu, Sijia Cui, Jinghan Zhu, Xinyu Zhan, Rongkun Li, Wei Tang, Jichao Wu, Ziying Wang, Mei Wang, Xiaojie Wang, Yan Zhang, Min Liu, Yusheng Xie, Yu Sun, Fan Yi
Senescent cells have long been associated with deleterious effects in aging-related pathologies, although recent studies have uncovered their beneficial roles in certain contexts such as wound healing. We have found that hepatic stellate cells (HSCs) undergo senescence within two days after 2/3 partial hepatectomy (PHx) in young (2-3 month-old) mice, and elimination of these senescent cells by the senolytic drug ABT263 or using a genetic mouse model impairs liver regeneration. Senescent HSCs secrete IL-6 and CXCR2 ligands as part of the senescence-associated secretory phenotype (SASP), which induces multiple signaling pathways to stimulate liver regeneration. IL-6 activates STAT3, induces YAP activation through SRC family kinases, and synergizes with CXCL2 to activate ERK1/2 to stimulate hepatocyte proliferation. The administration of either IL-6 or CXCL2 partially restores liver regeneration in mice with senescent cell elimination, and the combination of both fully restores liver weight recovery. Furthermore, the matricellular protein CCN1/CYR61 is rapidly elevated in response to PHx and induces HSC senescence. Knock-in mice expressing a mutant CCN1 unable to bind integrin α6β1 are deficient in senescent cells and liver regeneration after PHx. Thus, HSC senescence, largely induced by CCN1, is a programmed response to PHx and plays a critical role in liver regeneration through signaling pathways activated by IL-6 and ligands of CXCR2.
Naiyuan Cheng, Ki-Hyun Kim, Lester F. Lau
Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation as well as immune and vascular signaling by protease activated receptors (PARs). Here, we show that endothelial EPCRPAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCRPAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR−PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.
Magdalena L. Bochenek, Rajinikanth Gogiraju, Stefanie Großmann, Janina Krug, Jennifer Orth, Sabine Reyda, George S. Georgiadis, Henri Spronk, Stavros Konstantinides, Thomas Münzel, John H. Griffin, Philipp S. Wild, Christine Espinola-Klein, Wolfram Ruf, Katrin Schäfer
Chikungunya virus (CHIKV) is a re-emerging mosquito-borne alphavirus responsible for numerous outbreaks. Chikungunya can cause debilitating acute and chronic disease. Thus, the development of a safe and effective CHIKV vaccine is an urgent global health priority.This study evaluated the effectiveness of the live-attenuated CHIKV vaccine VLA1553 against WT CHIKV infection by using passive transfer of sera from vaccinated volunteers to non-human primates (NHP) subsequently exposed to WT CHIKV and established a serological surrogate of protection. We demonstrated that human VLA1553 sera transferred to NHPs conferred complete protection from CHIKV viremia and fever after challenge with homologous WT CHIKV. In addition, serum transfer protected animals from other CHIKV associated clinical symptoms and from CHIKV persistence in tissue. Based on this passive transfer study, a 50% micro plaque reduction neutralization test titer of ≥150 was determined as a surrogate of protection which was supported by analysis of samples from a sero-epidemiological study.In conclusion, considering the unfeasibility of an efficacy trial due to the unpredictability and explosive, rapidly moving nature of chikungunya outbreaks, the definition of a surrogate of protection for VLA1553 is an important step towards vaccine licensure to reduce the medical burden caused by chikungunya.
Pierre Roques, Andrea Fritzer, Nathalie Dereuddre-Bosquet, Nina Wressnigg, Romana Hochreiter, Laetitia Bossevot, Quentin Pascal, Fabienne Guehenneux, Annegret Bitzer, Irena Corbic Ramljak, Roger Le Grand, Urban Lundberg, Andreas Meinke
Familial exudative vitreoretinopathy (FEVR) is a hereditary disorder that can cause vision loss. The CTNND1 gene encodes a cellular adhesion protein p120-catenin (p120), which is essential for vascularization, yet the function of p120 in postnatal physiological angiogenesis remains unclear. Here, we applied whole-exome sequencing (WES) on 140 probands of FEVR families and identified three candidate variants in the human CTNND1 gene. We performed inducible deletion of Ctnnd1 in the postnatal mouse endothelial cells (ECs) and observed typical phenotypes of FEVR. Immunofluorescence of retina flat mounts also revealed immune responses, including reactive astrogliosis and microgliosis accompanied by abnormal Vegfa expression. Using an unbiased proteomics analysis in combination with in vivo or in vitro approaches, we propose that p120 is critical for the integrity of cadherin/catenin complex, and that p120 activates Wnt signaling activity by protecting β-catenin from Gsk3β-ubiquitin-guided degradation. Treatment of CTNND1-depleted HRECs with Gsk3β inhibitors LiCl or CHIR-99021 successfully enhanced cell proliferation by preventing β-catenin from degradation. Moreover, LiCl treatment increased vessel density in Ctnnd1-deficient mouse retinas. Functional analysis also revealed that variants in CTNND1 cause FEVR by compromising the expression of adherens junctions (AJs) and Wnt signaling activity. Additionally, genetic interactions between p120 and β-catenin or α-catenin revealed by double heterozygous deletion in mice further confirmed that p120 regulates vascular development through the Wnt/Cadherin axis. Together, we propose that CTNND1 is a novel candidate gene associated with FEVR, and that variants in CTNND1 can cause FEVR through the Wnt/Cadherin axis.
Mu Yang, Shujin Li, Li Huang, Rulian Zhao, Erkuan Dai, Xiaoyan Jiang, Yunqi He, Jinglin Lu, Li Peng, Wenjing Liu, Zhaotian Zhang, Dan Jiang, Yi Zhang, Zhilin Jiang, Yeming Yang, Peiquan Zhao, Xianjun Zhu, Xiaoyan Ding, Zhenglin Yang
Cardiac fibrosis, a primary contributor to heart failure (HF) and sudden death, is considered an important target for HF therapy. However, the signaling pathways that govern cardiac fibroblast (CF) function during cardiac fibrosis have not been fully elucidated. Here, we found that a functionally unannotated human myocardial infarction (MI) associated gene, family with sequence similarity 114 member A1 (FAM114A1), is induced in failing human and mouse hearts compared to non-failing hearts. Homozygous knockout of Fam114a1 (Fam114a1–/–) in the mouse genome reduces cardiac hypertrophy and fibrosis while significantly restores cardiac function in angiotensin (Ang) II- and MI-induced HF mouse models. Fam114a1 deletion antagonizes Ang II-induced inflammation and oxidative stress. Using isolated mouse primary CFs in wild type and Fam114a1–/– mice, we found that FAM114A1 is a critical autonomous factor for CF proliferation, activation, and migration. We discovered that FAM114A1 interacts with angiotensin receptor-associated protein (AGTRAP) and regulates the expression of angiotensin type 1 receptor (AT1R) and downstream Ang II signaling transduction, and subsequently influences pro-fibrotic response. Using RNA-Seq in mouse primary CFs, we identified differentially expressed genes, including extracellular matrix proteins such as Adamts15. RNAi-mediated inactivation of Adamts15 attenuates CF activation and collagen deposition. Our results indicate that FAM114A1 regulates Ang II signaling and downstream pro-fibrotic and pro-inflammatory gene expression, thereby activating cardiac fibroblasts and augmenting pathological cardiac remodeling. These findings provide novel insights into the regulation of cardiac fibrosis and identify FAM114A1 as a new therapeutic target for the treatment of cardiac disease.
Kadiam C. Venkata Subbaiah, Jiangbin Wu, Wai Hong Wilson Tang, Peng Yao
Vitiligo is an autoimmune skin disease characterized by the destruction of melanocytes by autoreactive CD8+ T cells. Melanocyte destruction in active vitiligo is mediated by CD8+ T cells but why white patches in stable disease persist is poorly understood. The interaction between immune cells, melanocytes, and keratinocytes in situ in human skin has been difficult to study due to the lack of proper tools. We combine non-invasive multiphoton microscopy (MPM) imaging and single-cell RNA sequencing (scRNA-seq) to identify subpopulations of keratinocytes in stable vitiligo patients. We show that, as compared to non-lesional skin, these keratinocytes are enriched in lesional vitiligo skin and shift their energy utilization towards oxidative phosphorylation. Systematic investigation of cell-cell communication networks show that this small population of keratinocyte secrete CXCL9 and CXCL10 to potentially drive vitiligo persistence. Pseudotemporal dynamics analyses predict an alternative differentiation trajectory that generates this new population of keratinocytes in vitiligo skin. Further MPM imaging of patients undergoing punch grafting treatment showed that keratinocytes favoring oxidative phosphorylation persist in non-responders but normalize in responders. In summary, we couple advanced imaging with transcriptomics and bioinformatics to discover cell-cell communication networks and keratinocyte cell states that can perpetuate inflammation and prevent repigmentation.
Jessica Shiu, Lihua Zhang, Griffin Lentsch, Jessica L. Flesher, Suoqin Jin, Christopher M. Polleys, Seong Jin Jo, Craig Mizzoni, Pezhman Mobasher, Jasmine Kwan, Francisca Rius-Diaz, Bruce J. Tromberg, Irene Georgakoudi, Qing Nie, Mihaela Balu, Anand K. Ganesan
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