Striated preferentially expressed protein kinase (SPEG), a myosin light chain kinase, is mutated in centronuclear myopathy (CNM) and/or dilated cardiomyopathy. No precise therapies are available for this disorder, and gene replacement therapy is not a feasible option due to the large size of SPEG. We evaluated the potential of dynamin-2 (DNM2) reduction as a potential therapeutic strategy because it has been shown to revert muscle phenotypes in mouse models of CNM caused by MTM1, DNM2, and BIN1 mutations. We determined that SPEG-β interacted with DNM2, and SPEG deficiency caused an increase in DNM2 levels. The DNM2 reduction strategy in Speg-KO mice was associated with an increase in life span, body weight, and motor performance. Additionally, it normalized the distribution of triadic proteins, triad ultrastructure, and triad number and restored phosphatidylinositol-3-phosphate levels in SPEG-deficient skeletal muscles. Although DNM2 reduction rescued the myopathy phenotype, it did not improve cardiac dysfunction, indicating a differential tissue-specific function. Combining DNM2 reduction with other strategies may be needed to target both the cardiac and skeletal defects associated with SPEG deficiency. DNM2 reduction should be explored as a therapeutic strategy against other genetic myopathies (and dystrophies) associated with a high level of DNM2.
Qifei Li, Jasmine Lin, Jeffrey J. Widrick, Shiyu Luo, Gu Li, Yuanfan Zhang, Jocelyn Laporte, Mark A. Perrella, Xiaoli Liu, Pankaj B. Agrawal
Angiopoietin-2 (Ang-2) is a key mediator of vascular disease during sepsis, and elevated plasma levels of Ang-2 are associated with organ injury scores and poor clinical outcomes. We have previously observed that biomarkers of endothelial glycocalyx (EG) damage correlate with plasma Ang-2 levels, suggesting a potential mechanistic linkage between EG injury and Ang-2 expression during states of systemic inflammation. However, the cell signaling mechanisms regulating Ang-2 expression following EG damage are unknown. In the current study, we determined the temporal associations between plasma heparan sulfate (HS) levels as a marker of EG erosion and plasma Ang-2 levels in children with sepsis and in mouse models of sepsis. Second, we evaluated the role of shear stress–mediated 5′-adenosine monophosphate–activated protein kinase (AMPK) signaling in Ang-2 expression following enzymatic HS cleavage from the surface of human primary lung microvascular endothelial cells (HLMVECs). We found that plasma HS levels peaked before plasma Ang-2 levels in children and mice with sepsis. Further, we discovered that impaired AMPK signaling contributed to increased Ang-2 expression following HS cleavage from flow-conditioned HLMVECs, establishing a paradigm by which Ang-2 may be upregulated during sepsis.
Robert P. Richter, Amit R. Ashtekar, Lei Zheng, Danielle Pretorius, Tripathi Kaushlendra, Ralph D. Sanderson, Amit Gaggar, Jillian R. Richter
Ciliopathies are a class of genetic diseases resulting in cilia dysfunction in multiple organ systems, including the olfactory system. Currently, there are no available curative treatments for olfactory dysfunction and other symptoms in ciliopathies. The loss or shortening of olfactory cilia, as seen in multiple mouse models of the ciliopathy Bardet–Biedl syndrome (BBS), results in olfactory dysfunction. However, the underlying mechanism of the olfactory cilia reduction is unknown, thus limiting the development of therapeutic approaches for BBS and other ciliopathies. Here, we demonstrated that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a phosphoinositide typically excluded from olfactory cilia, aberrantly redistributed into the residual cilia of BBS mouse models, which caused F-actin ciliary infiltration. Importantly, PI(4,5)P2 and F-actin were necessary for olfactory cilia shortening. Using a gene therapeutic approach, the hydrolyzation of PI(4,5)P2 by overexpression of inositol polyphosphate-5-phosphatase E (INPP5E) restored cilia length and rescued odor detection and odor perception in BBS. Together, our data indicate that PI(4,5)P2/F-actin–dependent cilia disassembly is a common mechanism contributing to the loss of olfactory cilia in BBS and provide valuable pan-therapeutic intervention targets for the treatment of ciliopathies.
Chao Xie, Julien C. Habif, Kirill Ukhanov, Cedric R. Uytingco, Lian Zhang, Robert J. Campbell, Jeffrey R. Martens
NLRP3 inflammasome and IFN-stimulated gene (ISG) induction are key biological drivers of ineffective hematopoiesis and inflammation in myelodysplastic syndromes (MDSs). Gene mutations involving mRNA splicing and epigenetic regulatory pathways induce inflammasome activation and myeloid lineage skewing in MDSs through undefined mechanisms. Using immortalized murine hematopoietic stem and progenitor cells harboring these somatic gene mutations and primary MDS BM specimens, we showed accumulation of unresolved R-loops and micronuclei with concurrent activation of the cytosolic sensor cyclic GMP-AMP synthase. Cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) signaling caused ISG induction, NLRP3 inflammasome activation, and maturation of the effector protease caspase-1. Deregulation of RNA polymerase III drove cytosolic R-loop generation, which upon inhibition, extinguished ISG and inflammasome response. Mechanistically, caspase-1 degraded the master erythroid transcription factor, GATA binding protein 1, provoking anemia and myeloid lineage bias that was reversed by cGAS inhibition in vitro and in Tet2–/– hematopoietic stem and progenitor cell–transplanted mice. Together, these data identified a mechanism by which functionally distinct mutations converged upon the cGAS/STING/NLRP3 axis in MDS, directing ISG induction, pyroptosis, and myeloid lineage skewing.
Amy F. McLemore, Hsin-An Hou, Benjamin S. Meyer, Nghi B. Lam, Grace A. Ward, Amy L. Aldrich, Matthew A. Rodrigues, Alexis Vedder, Ling Zhang, Eric Padron, Nicole D. Vincelette, David A. Sallman, Omar Abdel-Wahab, Alan F. List, Kathy L. McGraw
Current treatments fail to modify the underlying pathophysiology and disease progression of chronic obstructive pulmonary disease (COPD), necessitating alternative therapies. Here, we show that COPD subjects have increased IL-36γ and decreased IL-36 receptor antagonist (IL-36Ra) in bronchoalveolar and nasal fluid compared with control subjects. IL-36γ is derived from small airway epithelial cells (SAEC) and is further induced by a viral mimetic, whereas IL-36Ra is derived from macrophages. IL-36γ stimulates release of the neutrophil chemoattractants CXCL1 and CXCL8, as well as elastolytic matrix metalloproteinases (MMPs) from small airway fibroblasts (SAF). Proteases released from COPD neutrophils cleave and activate IL-36γ, thereby perpetuating IL-36 inflammation. Transfer of culture media from SAEC to SAF stimulated release of CXCL1, which was inhibited by exogenous IL-36Ra. The use of a therapeutic antibody that inhibits binding to the IL-36R attenuated IL-36γ–driven inflammation and cellular crosstalk. We have demonstrated a mechanism for the amplification and propagation of neutrophilic inflammation in COPD and have shown that blocking this cytokine family via a IL-36R neutralizing antibody could be a promising therapeutic strategy in the treatment of COPD.
Jonathan R. Baker, Peter S. Fenwick, Carolin K. Koss, Harriet B. Owles, Sarah L. Elkin, Jay Fine, Matthew Thomas, Karim C. El Kasmi, Peter J. Barnes, Louise E. Donnelly
Disrupted liver regeneration following hepatectomy represents an “undruggable” clinical challenge associated with poor patient outcomes. Yes-associated protein (YAP), a transcriptional coactivator that is repressed by the Hippo pathway, is instrumental in liver regeneration. We have previously described an alternative, Hippo-independent mechanism of YAP activation mediated by downregulation of protein tyrosine phosphatase nonreceptor type 11 (PTPN11, also known as SHP2) inhibition. Herein, we examined the effects of YAP activation with a selective SHP1/SHP2 inhibitor, NSC-87877, on liver regeneration in murine partial hepatectomy models. In our studies, NSC-87877 led to accelerated hepatocyte proliferation, improved liver regeneration, and decreased markers of injury following partial hepatectomy. The effects of NSC-87877 were lost in mice with hepatocyte-specific Yap/Taz deletion, and this demonstrated dependence on these molecules for the enhanced regenerative response. Furthermore, administration of NSC-87877 to murine models of nonalcoholic steatohepatitis was associated with improved survival and decreased markers of injury after hepatectomy. Evaluation of transcriptomic changes in the context of NSC-87877 administration revealed reduction in fibrotic signaling and augmentation of cell cycle signaling. Cytoprotective changes included downregulation of Nr4a1, an apoptosis inducer. Collectively, the data suggest that SHP2 inhibition induces a pro-proliferative and cytoprotective enhancement of liver regeneration dependent on YAP.
Ryan D. Watkins, EeeLN H. Buckarma, Jennifer L. Tomlinson, Chantal E. McCabe, Jennifer A. Yonkus, Nathan W. Werneburg, Rachel L. Bayer, Patrick P. Starlinger, Keith D. Robertson, Chen Wang, Gregory J. Gores, Rory L. Smoot
Integrins — the principal extracellular matrix (ECM) receptors of the cell — promote cell adhesion, migration, and proliferation, which are key events for cancer growth and metastasis. To date, most integrin-targeted cancer therapeutics have disrupted integrin-ECM interactions, which are viewed as critical for integrin functions. However, such agents have failed to improve cancer patient outcomes. We show that the highly expressed integrin β1 subunit is required for lung adenocarcinoma development in a carcinogen-induced mouse model. Likewise, human lung adenocarcinoma cell lines with integrin β1 deletion failed to form colonies in soft agar and tumors in mice. Mechanistically, we demonstrate that these effects do not require integrin β1–mediated adhesion to ECM but are dependent on integrin β1 cytoplasmic tail-mediated activation of focal adhesion kinase (FAK). These studies support a critical role for integrin β1 in lung tumorigenesis that is mediated through constitutive, ECM binding–independent signaling involving the cytoplasmic tail.
Scott M. Haake, Erin J. Plosa, Jonathan A. Kropski, Lindsay A. Venton, Anupama Reddy, Fabian Bock, Betty T. Chang, Allen J. Luna, Kateryna Nabukhotna, Zhi-Qi Xu, Rebecca A. Prather, Sharon Lee, Harikrishna Tanjore, Vasiliy V. Polosukhin, Olga M. Viquez, Angela Jones, Wentian Luo, Matthew H. Wilson, W. Kimryn Rathmell, Pierre P. Massion, Ambra Pozzi, Timothy S. Blackwell, Roy Zent
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 the 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 with 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 neurotoxic 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 with 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
Circulating monocytes have emerged as key regulators of the neuroinflammatory milieu in a number of neuropathological disorders. Ephrin type A receptor 4 (Epha4) receptor tyrosine kinase, a prominent axon guidance molecule, has recently been implicated in the regulation of neuroinflammation. Using a mouse model of brain injury and a GFP BM chimeric approach, we found neuroprotection and a lack of significant motor deficits marked by reduced monocyte/macrophage cortical infiltration and an increased number of arginase-1+ cells in the absence of BM-derived Epha4. This was accompanied by a shift in monocyte gene profile from pro- to antiinflammatory that included increased Tek (Tie2 receptor) expression. Inhibition of Tie2 attenuated enhanced expression of M2-like genes in cultured Epha4-null monocytes/macrophages. In Epha4-BM–deficient mice, cortical-isolated GFP+ monocytes/macrophages displayed a phenotypic shift from a classical to an intermediate subtype, which displayed reduced Ly6chi concomitant with increased Ly6clo- and Tie2-expressing populations. Furthermore, clodronate liposome–mediated monocyte depletion mimicked these effects in WT mice but resulted in attenuation of phenotype in Epha4-BM–deficient mice. This demonstrates that monocyte polarization not overall recruitment dictates neural tissue damage. Thus, coordination of monocyte proinflammatory phenotypic state by Epha4 is a key regulatory step mediating brain injury.
Elizabeth A. Kowalski, Eman Soliman, Colin Kelly, Erwin Kristobal Gudenschwager Basso, John Leonard, Kevin J. Pridham, Jing Ju, Alison Cash, Amanda Hazy, Caroline de Jager, Alexandra M. Kaloss, Hanzhang Ding, Raymundo D. Hernandez, Gabe Coleman, Xia Wang, Michelle L. Olsen, Alicia M. Pickrell, Michelle H. Theus
Older people exhibit dysregulated innate immunity to respiratory viral infections, including influenza and SARS-CoV-2, and show an increase in morbidity and mortality. Nanoparticles are a potential practical therapeutic that could reduce exaggerated innate immune responses within the lungs during viral infection. However, such therapeutics have not been examined for effectiveness during respiratory viral infection, particular in aged hosts. Here, we employed a lethal model of influenza viral infection in vulnerable aged mice to examine the ability of biodegradable, cargo-free nanoparticles, designated ONP-302, to resolve innate immune dysfunction and improve outcomes during infection. We administered ONP-302 via i.v. injection to aged mice at day 3 after infection, when the hyperinflammatory innate immune response was already established. During infection, we found that ONP-302 treatment reduced the numbers of inflammatory monocytes within the lungs and increased their number in both the liver and spleen, without impacting viral clearance. Importantly, cargo-free nanoparticles reduced lung damage, reduced histological lung inflammation, and improved gas exchange and, ultimately, the clinical outcomes in influenza-infected aged mice. In conclusion, ONP-302 improves outcomes in influenza-infected aged mice. Thus, our study provides information concerning a practical therapeutic, which, if translated clinically, could improve disease outcomes for vulnerable older patients suffering from respiratory viral infections.
William J. Kelley, Kathleen M. Wragg, Judy Chen, Tushar Murthy, Qichen Xu, Michael T. Boyne, Joseph R. Podojil, Adam Elhofy, Daniel R. Goldstein
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