Understanding the circuits that promote an efficient resolution of inflammation is crucial to deciphering the molecular and cellular processes required to promote tissue repair. Macrophages play a central role in the regulation of inflammation, resolution, and repair/regeneration. Using a model of skeletal muscle injury and repair, herein we identified annexin A1 (AnxA1) as the extracellular trigger of macrophage skewing toward a pro-reparative phenotype. Brought into the injured tissue initially by migrated neutrophils, and then overexpressed in infiltrating macrophages, AnxA1 activated FPR2/ALX receptors and the downstream AMPK signaling cascade, leading to macrophage skewing, dampening of inflammation, and regeneration of muscle fibers. Mice lacking AnxA1 in all cells or only in myeloid cells displayed a defect in this reparative process. In vitro experiments recapitulated these properties, with AMPK-null macrophages lacking AnxA1-mediated polarization. Collectively, these data identified the AnxA1/FPR2/AMPK axis as an important pathway in skeletal muscle injury regeneration.
Simon McArthur, Gaëtan Juban, Thomas Gobbetti, Thibaut Desgeorges, Marine Theret, Julien Gondin, Juliana E. Toller-Kawahisa, Chris P. Reutelingsperger, Bénédicte Chazaud, Mauro Perretti, Rémi Mounier
Mutations in APC promote colorectal cancer (CRC) progression through uncontrolled WNT signaling. Patients with desmoplastic CRC have a significantly worse prognosis and do not benefit from chemotherapy, but the mechanisms underlying the differential responses of APC-mutant CRCs to chemotherapy are not well understood. We report that expression of the transcription factor prospero homeobox 1 (PROX1) was reduced in desmoplastic APC-mutant human CRCs. In genetic Apc-mutant mouse models, loss of Prox1 promoted the growth of desmoplastic, angiogenic, and immunologically silent tumors through derepression of Mmp14. Although chemotherapy inhibited Prox1-proficient tumors, it promoted further stromal activation, angiogenesis, and invasion in Prox1-deficient tumors. Blockade of vascular endothelial growth factor A (VEGFA) and angiopoietin-2 (ANGPT2) combined with CD40 agonistic antibodies promoted antiangiogenic and immunostimulatory reprogramming of Prox1-deficient tumors, destroyed tumor fibrosis, and unleashed T cell–mediated killing of cancer cells. These results pinpoint the mechanistic basis of chemotherapy-induced hyperprogression and illustrate a therapeutic strategy for chemoresistant and desmoplastic CRCs.
Simone Ragusa, Borja Prat-Luri, Alejandra González-Loyola, Sina Nassiri, Mario Leonardo Squadrito, Alan Guichard, Sabrina Cavin, Nikolce Gjorevski, David Barras, Giancarlo Marra, Matthias P. Lutolf, Jean Perentes, Emily Corse, Roberta Bianchi, Laureline Wetterwald, Jaeryung Kim, Guillermo Oliver, Mauro Delorenzi, Michele De Palma, Tatiana V. Petrova
Despite advancements in targeting the immune checkpoints program cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), and cytotoxic T lymphocyte–associated protein 4 (CTLA-4) for cancer immunotherapy, a large number of patients and cancer types remain unresponsive. Current immunotherapies focus on modulating an antitumor immune response by directly or indirectly expanding antitumor CD8 T cells. A complementary strategy might involve inhibition of Tregs that otherwise suppress antitumor immune responses. Here, we sought to identify functional immune molecules preferentially expressed on tumor-infiltrating Tregs. Using genome-wide RNA-Seq analysis of purified Tregs sorted from multiple human cancer types, we identified a conserved Treg immune checkpoint signature. Using immunocompetent murine tumor models, we found that antibody-mediated depletion of 4-1BB–expressing cells (4-1BB is also known as TNFRSF9 or CD137) decreased tumor growth without negatively affecting CD8 T cell function. Furthermore, we found that the immune checkpoint 4-1BB had a high selectivity for human tumor Tregs and was associated with worse survival outcomes in patients with multiple tumor types. Thus, antibody-mediated depletion of 4-1BB–expressing Tregs represents a strategy with potential activity across cancer types.
Zachary T. Freeman, Thomas R. Nirschl, Daniel H. Hovelson, Robert J. Johnston, John J. Engelhardt, Mark J. Selby, Christina M. Kochel, Ruth Y. Lan, Jingyi Zhai, Ali Ghasemzadeh, Anuj Gupta, Alyza M. Skaist, Sarah J. Wheelan, Hui Jiang, Alexander T. Pearson, Linda A. Snyder, Alan J. Korman, Scott A. Tomlins, Srinivasan Yegnasubramanian, Charles G. Drake
A single subanesthetic dose of ketamine, an NMDA receptor (NMDAR) antagonist, produces rapid and sustained antidepressant actions in depressed patients, addressing a major unmet need for the treatment of mood disorders. Ketamine produces a rapid increase in extracellular glutamate and synaptic formation in the prefrontal cortex, but the initial cellular trigger that initiates this increase and ketamine’s behavioral actions has not been identified. To address this question, we used a combination of viral shRNA and conditional mutation to produce cell-specific knockdown or deletion of a key NMDAR subunit, GluN2B, implicated in the actions of ketamine. The results demonstrated that the antidepressant actions of ketamine were blocked by GluN2B-NMDAR knockdown on GABA (Gad1) interneurons, as well as subtypes expressing somatostatin (Sst) or parvalbumin (Pvalb), but not glutamate principle neurons in the medial prefrontal cortex (mPFC). Further analysis of GABA subtypes showed that cell-specific knockdown or deletion of GluN2B in Sst interneurons blocked or occluded the antidepressant actions of ketamine and revealed sex-specific differences that are associated with excitatory postsynaptic currents on mPFC principle neurons. These findings demonstrate that GluN2B-NMDARs on GABA interneurons are the initial cellular trigger for the rapid antidepressant actions of ketamine and show sex-specific adaptive mechanisms to GluN2B modulation.
Danielle M. Gerhard, Santosh Pothula, Rong-Jian Liu, Min Wu, Xiao-Yuan Li, Matthew J. Girgenti, Seth R. Taylor, Catharine H. Duman, Eric Delpire, Marina Picciotto, Eric S. Wohleb, Ronald S. Duman
Immune response to therapeutic enzymes poses a detriment to patient safety and treatment outcome. Enzyme replacement therapy (ERT) is a standard therapeutic option for some types of mucopolysaccharidoses, including Morquio A syndrome caused by N-acetylgalactosamine-6-sulfate sulfatase (GALNS) deficiency. Current protocols tolerize patients using cytotoxic immunosuppressives, which can cause adverse effects. Here we show development of tolerance in Morquio A mice via oral delivery of peptide or GALNS for 10 days prior to ERT. Our results show that using an immunodominant peptide (I10) or the complete GALNS enzyme to orally induce tolerance to GALNS prior to ERT resulted in several improvements to ERT in mice: (a) decreased splenocyte proliferation after in vitro GALNS stimulation, (b) modulation of the cytokine secretion profile, (c) decrease in GALNS-specific IgG or IgE in plasma, (d) decreased GAG storage in liver, and (e) fewer circulating immune complexes in plasma. This model could be extrapolated to other lysosomal storage disorders in which immune response hinders ERT.
Angela C. Sosa, Barbara Kariuki, Qi Gan, Alan P. Knutsen, Clifford J. Bellone, Miguel A. Guzmán, Luis A. Barrera, Shunji Tomatsu, Anil K. Chauhan, Eric Armbrecht, Adriana M. Montaño
Cortical hyperexcitability and mislocalization of the RNA-binding protein TDP43 are highly conserved features in amyotrophic lateral sclerosis (ALS). Nevertheless, the relationship between these phenomena remains poorly defined. Here, we showed that hyperexcitability recapitulates TDP43 pathology by upregulating shortened TDP43 (sTDP43) splice isoforms. These truncated isoforms accumulated in the cytoplasm and formed insoluble inclusions that sequestered full-length TDP43 via preserved N-terminal interactions. Consistent with these findings, sTDP43 overexpression was toxic to mammalian neurons, suggesting neurodegeneration arising from complementary gain- and loss-of-function mechanisms. In humans and mice, sTDP43 transcripts were enriched in vulnerable motor neurons, and we observed a striking accumulation of sTDP43 within neurons and glia of ALS patients. Collectively, these studies uncover a pathogenic role for alternative TDP43 isoforms in ALS, and implicate sTDP43 as a key contributor to the susceptibility of motor neurons in this disorder.
Kaitlin Weskamp, Elizabeth M. Tank, Roberto Miguez, Jonathon P. McBride, Nicolás B. Gómez, Matthew White, Ziqiang Lin, Carmen Moreno Gonzalez, Andrea Serio, Jemeen Sreedharan, Sami J. Barmada
Influenza A virus (IAV) is among the most common causes of pneumonia-related death worldwide. Pulmonary epithelial cells are the primary target for viral infection and replication and respond by releasing inflammatory mediators that recruit immune cells to mount the host response. Severe lung injury and death during IAV infection result from an exuberant host inflammatory response. The linear ubiquitin assembly complex (LUBAC), composed of SHARPIN, HOIL-1L, and HOIP, is a critical regulator of NF-κB–dependent inflammation. Using mice with lung epithelial–specific deletions of HOIL-1L or HOIP in a model of IAV infection, we provided evidence that, while a reduction in the inflammatory response was beneficial, ablation of the LUBAC-dependent lung epithelial–driven response worsened lung injury and increased mortality. Moreover, we described a mechanism for the upregulation of HOIL-1L in infected and noninfected cells triggered by the activation of type I IFN receptor and mediated by IRF1, which was maladaptive and contributed to hyperinflammation. Thus, we propose that lung epithelial LUBAC acts as a molecular rheostat that could be selectively targeted to modulate the immune response in patients with severe IAV-induced pneumonia.
Patricia L. Brazee, Luisa Morales-Nebreda, Natalia D. Magnani, Joe G.N. Garcia, Alexander V. Misharin, Karen M. Ridge, G.R. Scott Budinger, Kazuhiro Iwai, Laura A. Dada, Jacob I. Sznajder
Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28–36), a neutral endopeptidase–generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28–36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10–dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28–36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC–/– cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28–36) and demonstrate that the ability of GLP-1(28–36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28–36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.
M. Ahsan Siraj, Dhanwantee Mundil, Sanja Beca, Abdul Momen, Eric A. Shikatani, Talat Afroze, Xuetao Sun, Ying Liu, Siavash Ghaffari, Warren Lee, Michael B. Wheeler, Gordon Keller, Peter Backx, Mansoor Husain
Posttranslational modifications (PTMs) are common among proteins that aggregate in neurodegenerative disease, yet how PTMs impact the aggregate conformation and disease progression remains unclear. By engineering knockin mice expressing prion protein (PrP) lacking 2 N-linked glycans (Prnp180Q/196Q), we provide evidence that glycans reduce spongiform degeneration and hinder plaque formation in prion disease. Prnp180Q/196Q mice challenged with 2 subfibrillar, non–plaque-forming prion strains instead developed plaques highly enriched in ADAM10-cleaved PrP and heparan sulfate (HS). Intriguingly, a third strain composed of intact, glycophosphatidylinositol-anchored (GPI-anchored) PrP was relatively unchanged, forming diffuse, HS-deficient deposits in both the Prnp180Q/196Q and WT mice, underscoring the pivotal role of the GPI-anchor in driving the aggregate conformation and disease phenotype. Finally, knockin mice expressing triglycosylated PrP (Prnp187N) challenged with a plaque-forming prion strain showed a phenotype reversal, with a striking disease acceleration and switch from plaques to predominantly diffuse, subfibrillar deposits. Our findings suggest that the dominance of subfibrillar aggregates in prion disease is due to the replication of GPI-anchored prions, with fibrillar plaques forming from poorly glycosylated, GPI-anchorless prions that interact with extracellular HS. These studies provide insight into how PTMs impact PrP interactions with polyanionic cofactors, and highlight PTMs as a major force driving the prion disease phenotype.
Alejandro M. Sevillano, Patricia Aguilar-Calvo, Timothy D. Kurt, Jessica A. Lawrence, Katrin Soldau, Thu H. Nam, Taylor Schumann, Donald P. Pizzo, Sofie Nyström, Biswa Choudhury, Hermann Altmeppen, Jeffrey D. Esko, Markus Glatzel, K. Peter R. Nilsson, Christina J. Sigurdson
Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and noninvasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to noninvasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell–derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine-learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate noninvasive cell therapy characterization can be achieved with QBAM and machine learning.
Nicholas J. Schaub, Nathan A. Hotaling, Petre Manescu, Sarala Padi, Qin Wan, Ruchi Sharma, Aman George, Joe Chalfoun, Mylene Simon, Mohamed Ouladi, Carl G. Simon Jr., Peter Bajcsy, Kapil Bharti
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