Excessive lipolysis in white adipose tissues (WAT) leads to insulin resistance (IR) and ectopic fat accumulation in insulin-sensitive tissues. However, the impact of Gi-coupled receptors in restraining adipocyte lipolysis through inhibition of cAMP production remained poorly elucidated. Given that the Gi-coupled P2Y13 receptor (P2Y13-R) is a purinergic receptor expressed in WAT, we investigated its role in adipocyte lipolysis and its effect on IR and metabolic dysfunction-associated steatotic liver disease (MASLD). In human, mRNA expression of P2Y13-R in WAT was negatively correlated to adipocytes lipolysis. In mice, adipocytes lacking P2Y13-R displayed higher intracellular cAMP levels, indicating impaired Gi signaling. Consistently, the absence of P2Y13-R was linked to increased lipolysis in adipocytes and WAT explants via hormone-sensitive lipase activation. Metabolic studies indicate that mice lacking P2Y13-R show a greater susceptibility to diet-induced IR, systemic inflammation, and MASLD compared to their wild-type counterparts. Assays conducted on precision-cut liver slices exposed to WAT conditioned medium and on liver-specific P2Y13-R knockdown mice suggested that P2Y13-R activity in WAT protects from hepatic steatosis, independently of liver P2Y13-R expression. In conclusion, our findings support the idea that targeting adipose P2Y13-R activity may represent a pharmacological strategy to prevent obesity-associated disorders, including type 2 diabetes and MASLD.
Thibaut Duparc, Emilia Gore, Guillaume Combes, Diane Beuzelin, Julie Pires Da Silva, Vanessa Bouguetoch, Marie-Adeline Marquès, Ana Velazquez, Nathalie Viguerie, Geneviève Tavernier, Peter Arner, Mikael Rydén, Dominique Langin, Nabil Sioufi, Mohamad Nasser, Cendrine Cabou, Souad Najib, Laurent O. Martinez
Allergic Airway Disease (AAD) is an example of type 2 inflammation which leads to chronic airway eosinophilia controlled by CD4 Th2 cells. Inflammation is reinforced by mast cells and basophils armed with allergen-specific IgE made by allergen-specific B2 B cells of the adaptive immune system. Little is known about how AAD is affected by innate B1 cells which produce natural antibodies (NAbs) that facilitate apoptotic cell clearance and detect damage and pathogen associated molecular patterns (DAMPS and PAMPS). We used transgenic mouse models lacking either B cells or NAbs in distinct mouse models of AAD, that require either DAMPS or PAMPS as the initial trigger for type 2 immunity. In a DAMP-induced allergic model, driven by alum and uric acid, mouse strains lacking B cells (CD19DTA), NAbs (IgHEL MD4), or all secreted antibodies (sIgm-/-Aid-/-), displayed significant reduction in both eosinophilia and Th2 priming compared to wild-type or Aid-/- mice lacking only germinal center dependent high-affinity class switched antibodies. Replenishing B-cell deficient mice with either unimmunized B1 B cells or NAbs during sensitization restored eosinophilia, suggesting NAbs are required for licensing antigen presenting cells to prime type 2 immunity. Conversely, PAMP-dependent type 2 priming to house dust mite or Aspergillus were not dependent on NAbs. This study reveals an underappreciated role of B1 B cell-generated natural antibodies in selectively driving DAMP-induced type-2 immunity.
Arlind B. Mara, Kavita Rawat, William T. King, Claudia V. Jakubzick
Studies on severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) have highlighted the crucial role of host proteases for viral replication and the immune response. The serine proteases furin and TMPRSS2 and lysosomal cysteine proteases were shown to facilitate virus entry by limited proteolytic processing of the spike (S) protein. While neutrophils are recruited to the lungs during COVID-19 pneumonia, little is known about the role of the neutrophil serine proteases (NSPs) cathepsin G (CatG), elastase (NE), and proteinase 3 (PR3) on SARS-CoV-2 entry and replication. Furthermore, the current paradigm is that NSPs may contribute to the pathogenesis of severe COVID-19. Here, we show that these proteases cleave the S protein at multiple sites and abrogate virus entry and replication in vitro. In mouse models, CatG significantly inhibited viral replication in the lung. Importantly, lung inflammation and pathology were increased in mice deficient in NE and/or CatG. These results reveal that NSPs contribute to innate defenses against SARS-CoV-2 infection via proteolytic inactivation of the S protein and that NE and CatG limit lung inflammation in vivo. We conclude that therapeutic interventions aiming to reduce the activity of NSPs may interfere with virus clearance and inflammation in COVID-19 patients.
Nathan G.F. Leborgne, Christelle Devisme, Nedim Kozarac, Inês Berenguer Veiga, Nadine Ebert, Aurélie Godel, Llorenç Grau-Roma, Melanie Scherer, Philippe Plattet, Volker Thiel, Gert Zimmer, Adriano Taddeo, Charaf Benarafa
Prior studies showed that polyQ-expanded AR is aberrantly acetylated and that deacetylation of the mutant AR by overexpression of NAD+-dependent sirtuin 1 (SIRT1) is protective in cell models of spinal and bulbar muscular atrophy (SBMA). Based on these observations and reduced NAD+ in muscles of SBMA mouse models, we tested the therapeutic potential of NAD+ restoration in vivo by treating post-symptomatic transgenic SBMA mice with the nicotinamide adenine dinucleotide (NAD+) precursor nicotinamide riboside (NR). NR supplementation failed to alter disease progression and had no effect on increasing NAD+ or ATP content in muscle, despite producing a modest increase of NAD+ in the spinal cord of SBMA mice. Metabolite and proteomic profiles of SBMA quadriceps muscles indicated alterations in several important energy-related pathways that utilize NAD+, in addition to the NAD salvage pathway, which is critical for NAD+ regeneration for use in cellular energy production. We also observed decreased mRNA levels of Nmrk2, which encodes a key kinase responsible for NR phosphorylation, allowing its utilization by the NAD salvage pathway. Together these data suggest a model in which NAD+ levels are significantly decreased in muscles of an SBMA mouse model and intransigent to NR supplementation due to decreased levels of Nmrk2.
Danielle DeBartolo, Frederick J. Arnold, Yuhong Liu, Elana Molotsky, Hsin-Yao Tang, Diane E. Merry
Mesenchymal stem cells, suffering from diverse gene hits, undergoes malignant transformation and aberrant osteochondral differentiation. Src homology region 2- (SH2-) containing protein tyrosine phosphatase 2 (SHP2), a non-receptor protein tyrosine phosphatase, regulates multicellular differentiation, proliferation, and transformation. However, the role of SHP2 in MSC fate determination remains unclear. Here, we showed that MSCs bearing the activating SHP2E76K mutation underwent malignant transformation into sarcoma stem-like cells (SSCs). We revealed that the SHP2E76K mutation in mouse MSCs led to hyperactive mitochondrial metabolism by activating mitochondrial complexes I and III. Inhibition of complexes I and III prevented hyperactive mitochondrial metabolism and malignant transformation of SHP2E76K MSCs. Mechanistically, we confirmed that SHP2 underwent liquid–liquid phase separation (LLPS) in SHP2E76K MSCs. SHP2 LLPS led to its dissociation from complexes I and III, causing their hyperactivation. Blockade of SHP2 LLPS by LLPS‒defective mutations or allosteric inhibitors suppressed complex I and III hyperactivation as well as malignant transformation of SHP2E76K MSCs. These findings reveal that complex I and III hyperactivation driven by SHP2 LLPS promotes malignant transformation of SHP2E76K MSCs and suggest that inhibition of SHP2 LLPS could be a potential therapeutic target for the treatment of activating SHP2‒associated cancers.
Chen Kan, Zhenya Tan, Liwei Liu, Bo Liu, Li Zhan, Jicheng Zhu, Xiaofei Li, Keqiong Lin, Jia Liu, Yakun Liu, Fan Yang, Mandy Wong, Siying Wang, Hong Zheng
Accumulation of sphingolipids, especially sphingosines, in the lysosomes is a key driver of several lysosomal storage diseases. The transport mechanism for sphingolipids from the lysosome remains unclear. Here, we identified SPNS1, which shares the highest homology to SPNS2 - a sphingosine-1-phosphate (S1P) transporter, functions as a transporter for lysolipids from the lysosome. We generated Spns1 knockout cells and mice and employed lipidomic and metabolomic approaches to reveal SPNS1 ligand identity. Global knockout of Spns1 caused embryonic lethality between E12.5-E13.5 and an accumulation of sphingosine, lysophosphatidylcholines (LPC) and lysophosphatidylethanolamines (LPE) in the fetal livers. Similarly, metabolomic analysis of livers from postnatal Spns1 knockout (Spns1-KO) mice presented an accumulation of sphingosines and lysoglycerophospholipids including LPC and LPE. Subsequently, biochemical assays showed that SPNS1 is required for LPC and sphingosine release from lysosomes. The accumulation of these lysolipids in the lysosomes of Spns1-KO mice affected liver functions and altered the PI3K-AKT signaling pathway. Furthermore, we identified three human siblings with a homozygous variant in the SPNS1 gene. These patients suffer from developmental delay, neurological impairment, intellectual disability, and exhibiting cerebellar hypoplasia. These results reveal a critical role of SPNS1 as a promiscuous lysolipid transporter in the lysosomes and link its physiological functions with lysosomal storage diseases.
Hoa T.T. Ha, SiYi Liu, Xuan T.A. Nguyen, Linh K. Vo, Nancy C.P. Leong, Dat T. Nguyen, Shivaranjani Balamurugan, Pei Yen Lim, YaJun Wu, Eunju Seong, Toan Q. Nguyen, Jeongah Oh, Markus R. Wenk, Amaury Cazenave-Gassiot, Zuhal Yapici, Wei-Yi Ong, Margit Burmeister, Long N. Nguyen
Programmed cell death protein 1 (PD-1), a coinhibitory T-cell checkpoint, is also expressed on macrophages (Mφ) in pathogen- or tumor-driven chronic inflammation. Increasing evidence underscores the importance of PD-1 on Mφ for dampening immune responses. However, the mechanism governing PD-1 expression in Mφ in chronic inflammation remains largely unknown. TGF-β1 (transforming growth factor-β1) is abundant within chronic inflammatory microenvironments. Here, based on public databases, significant positive correlations between PDCD1 and TGFB1 gene expression were observed in most human tumors. Of note, among immune infiltrates, Mφ as the predominant infiltrate expressed higher PDCD1 and TGFBR1/TGFBR2 genes. MC38 colon cancer and S. japonicum infection were used as experimental models for chronic inflammation. PD-1hi Mφ from chronic inflammatory tissues displayed an immunoregulatory pattern and expressed a higher level of TGF-β receptors. Either TGF-β1-neutralizing antibody administration or Mφ-specific Tgfbr1 knockdown largely reduced PD-1 expression on Mφ in animal models. We further demonstrated that TGF-β1 directly induced PD-1 expression on Mφ. Mechanistically, TGF-β1-induced PD-1 expression on Mφ was dependent on SMAD3 and STAT3, which formed a complex at the Pdcd1 promoter. Collectively, our study shows that Mφ adapt to chronic inflammation through TGF-β1-triggered cooperative SMAD3-STAT3 signaling that induces PD-1 expression and modulates Mφ function.
Zhigang Lei, Rui Tang, Yu Wu, Chenxu Mao, Weijie Xue, Junyao Shen, Jiaojiao Yu, Xiaohong Wang, Xin Qi, Chuan Wei, Lei Xu, Jifeng Zhu, Yalin Li, Xiujun Zhang, Chunyan Ye, Xiaojun Chen, Xiaojun Yang, Sha Zhou, Chuan Su
Compromised vascular integrity facilitates extravasation of cancer cells and promotes metastatic dissemination. CD93 has emerged as a target for anti-angiogenic therapy, but its importance for vascular integrity in metastatic cancers has not been evaluated. Here, we demonstrate that CD93 participates in maintaining the endothelial barrier and reducing metastatic dissemination. Primary melanoma growth was hampered in CD93-/- mice but metastatic dissemination was increased, associated with a disruption of adherens and tight junctions in tumor endothelial cells and elevated expression of matrix metalloprotease 9 (MMP9) at the metastatic site. CD93 directly interacted with vascular endothelial growth factor receptor 2 (VEGFR2) and its absence led to VEGF-induced hyper-phosphorylation of VEGFR2 in endothelial cells. Antagonistic-VEGFR2 antibody therapy rescued endothelial barrier function and reduced the metastatic burden in CD93-/- mice to wild-type levels. These findings reveal a key role of CD93 in maintaining vascular integrity, which has implications for pathological angiogenesis and endothelial barrier function in metastatic cancer.
Kalyani Vemuri, Beatriz de Alves Pereira, Patricia Fuenzalida, Yelin Subashi, Stefano Barbera, Luuk van Hooren, Marie Hedlund, Fredrik Pontén, Cecilia Lindskog, Anna-Karin Olsson, Roberta Lugano, Anna Dimberg
Myocardial ischemia/reperfusion (MI/R) injury is a major cause of adverse outcomes of revascularization following myocardial infarction. Anaerobic glycolysis during myocardial ischemia is well-studied, but the role of aerobic glycolysis during the early phase of reperfusion is incompletely understood. Lactylation of Histone H3 (H3) is an epigenetic indicator of the glycolytic switch. Heat shock protein A12A (HSPA12A) is an atypic member of the HSP70 family. In the present study, we report that during reperfusion following myocardial ischemia, HSPA12A was downregulated and aerobic glycolytic flux was decreased in cardiomyocytes. Notably, HSPA12A knockout in mice exacerbated MI/R-induced aerobic glycolysis decrease, cardiomyocyte death, and cardiac dysfunction. Gain- and loss-of-function studies demonstrated that HSPA12A was required to support cardiomyocyte survival upon hypoxia/reoxygenation (H/R) challenge, and that its protective effects were mediated by maintaining aerobic glycolytic homeostasis for H3 lactylation. Further analyses revealed that HSPA12A increased Smurf1-mediated Hif1α protein stability, thus increasing glycolytic gene expression to maintain appropriate aerobic glycolytic activity to sustain H3 lactylation during reperfusion, and ultimately improving cardiomyocyte survival to attenuate MI/R injury.
Wansu Yu, Qiuyue Kong, Surong Jiang, Yunfan Li, Zhaohe Wang, Qian Mao, Xiaojin Zhang, Qianhui Liu, Pengjun Zhang, Yuehua Li, Chuanfu Li, Zhengnian Ding, Li Liu
Allelic heterogeneity (AH) has been noted in truncational TTN (TTNtv)-associated dilated cardiomyopathy (DCM), i.e., mutations affecting A-band-encoding exons are pathogenic, but those affecting Z-disc-encoding exons are likely benign. The lack of an in vivo animal model that recapitulates AH hinders the deciphering of the underlying mechanism. Here, we explored zebrafish as a candidate vertebrate model by phenotyping a collection of zebrafish ttntv alleles. We noted that cardiac function and sarcomere structure are more severely disrupted in ttntv-A than in ttntv-Z homozygous embryos. Consistently, cardiomyopathy-like phenotypes were presented in ttntv-A but not ttntv-Z adult heterozygous mutants. The phenotypes observed in ttntv-A alleles were recapitulated in null mutants with the entire titin-encoding sequences removed. Defective autophagic flux, largely due to impaired autophagosome-lysosome fusion, was also only noted in ttntv-A but not ttntv-Z models. Moreover, we found that genetic manipulation of ulk1a restored autophagy flux and rescued cardiac dysfunction in ttntv-A animals. Together, our findings presented adult zebrafish as an in vivo animal model for studying AH in TTNtv DCM, demonstrated TTN loss-of-function sufficient to trigger ttntv DCM in zebrafish, and uncovered ulk1a as a potential therapeutic target gene for TTNtv DCM.
Ping Zhu, Jiarong Li, Feixiang Yan, Shahidul Islam, Xueying Lin, Xiaolei Xu
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