Although the involvement of the H3.3K27M mutation in Diffuse Midline Glioma tumorigenesis is now established, its role in their resistance to treatments and, therefore, in their fatal outcome remains poorly documented. Here, thanks to our models of H3.3K27M induction in pediatric glioma cells, we finally shed light on this crucial issue. Hence, we demonstrate here for the first time that H3.3K27M can increase cell radioresistance capabilities independently of TP53 alterations. Moreover, thanks to a drug library screening, we evidenced that this mutation can, depending on the cellular context, drastically modulate the response of these cells to different classes of compounds, thus paving the way for new therapeutic strategies. Altogether, our results provide here the proof that, beyond its role in tumorigenesis, the presence of H3.3K27M mutation by itself alters the response to treatments of pediatric glioma cells.High-grade gliomas represent the most lethal class of pediatric tumors, and their resistance to both radio- and chemotherapy is associated with a poor prognosis. Recurrent mutations affecting histone genes drive the tumorigenesis of some pediatric high-grade gliomas, and H3K27M mutations are notably characteristic of a subtype of gliomas called DMG (Diffuse Midline Gliomas). This dominant negative mutation impairs H3K27 trimethylation, leading to profound epigenetic modifications of genes expression. Even though this mutation was described as a driver event in tumorigenesis, its role in tumor cell resistance to treatments has not been deciphered so far. To tackle this issue, we expressed the H3.3K27M mutated histone in three initially H3K27-unmutated pediatric glioma cell lines, Res259, SF188, and KNS42. First, we validated these new H3.3K27M-expressing models at the molecular level and showed that K27M expression is associated with pleiotropic effects on the transcriptomic signature, largely dependent on cell context. We observed that the mutation triggered an increase in cell growth in Res259 and SF188 cells, associated with higher clonogenic capacities. Interestingly, we evidenced that the mutation confers an increased resistance to ionizing radiations in Res259 and KNS42 cells. Moreover, we showed that H3.3K27M mutation impacts the sensitivity of Res259 cells to specific drugs among a library of 80 anticancerous compounds. Altogether, these data highlight that, beyond its tumorigenic role, H3.3K27M mutation is strongly involved in pediatric glioma cells’ resistance to therapies, likely through transcriptomic reprogramming. Tumors of the central nervous system (CNS) are the most frequent solid tumors in children, representing around 25% of pediatric cancers . Among tDIPGs are almost exclusively pediatric and to date, the extrapolation of data obtained from adult glioma patients has proven insufficient to increase the patient’s survival . They arH3F3A gene (around 73% of cases), coding the H3.3 variant, but are also found in the HIST1H3B/C gene, coding for the H3.1 variant (around 26% of cases). Mutation in the H3.2 variant was also detected in a few tumors (<1% of cases). H3K27M mutations have a dominant-negative effect that results in a global loss of the H3K27me3 epigenetic marks in the genome [TP53 mutation and PDGFRA amplification [The discovery of the recurrent H3K27M somatic mutations in histone H3 genes provided new insights into the biology of DIPG ,10,11,12e genome . They wefication ,15,16. Afication .Of note, epigenetic alterations are common in pediatric HGG. Indeed, most H3K27 non-mutated DIPG and PFA (Posterior Fossa A) ependymomas display a similar epigenetic rewiring as Diffuse Midline Glioma H3K27M mutant, notably driven by EZHIP over-expression ,20,21,22H3F3A gene in pLGG and pHGG cell lines and assessed the consequences on epigenetic marks, gene expression, and biological processes, including cell growth and clonogenicity. Finally, we evaluated, for the first time, the specific impact of the mutation on the response to a wide panel of anticancer drugs but also to ionizing radiations, which remain the standard of care treatment in the clinic.Beyond these targets, to the best of our knowledge, the specific impact of H3K27M mutation in cell resistance to a wide range of therapies has not been elucidated. In that frame, we here evaluate the impact of inducing H3.3K27M mutation in both low- and high-grade pediatric glioma cells. We introduced the K27M-mutated MYC and CCND1 amplifications, KNS42 cells have a low-level copy number gain of PI3KCA locus and, Res259 cells harbor an amplification of PDGFRA and a deletion of CDKN2A/B. A more detailed phenotypic and molecular characterization of these cell lines was previously described by Bax and colleagues [2.Human pediatric high-grade glioma cell lines SF188 and KNS42  and low-grade glioma cell line Res259  were kindly provided by Dr Chris Jones . Among other genetic alterations, SF188 cells harbor lleagues . All cel6 cells was transfected using Cell Line NucleofectorTM Kit V  with 1µg of plasmid containing K27M mutated H3F3A gene fused with the mCherry gene, and bearing a resistance gene for Hygromycin B. As a control, cells were transfected with a similar plasmid containing the WT H3F3A gene. After 2 days, Hygromycin B was added at 400 µg/mL. After 2 weeks of selection, cells were sorted with an ARIA III SORP  flow cytometer using the 561 nm laser line and the emission filter 610/20.Cells were dissociated, collected and 1 × 10Cells were seeded in a chambered 8-well coverslip µ-Slide  at a density of 25,000 cells per well. After 24 to 48 h of incubation, cells were labeled with Hoechst  to visualize the cell nuclei. Cells were then observed by confocal laser scanning microscopy , using a Laser diode (405 nm) and a Laser line (561 nm), and the respective appropriate emission filters (445/50 and 605/70) with a 20× objective.1/1000. The secondary anti-rabbit antibody coupled with HRP  was diluted at 1/2000 in BSA buffer. Signals were revealed using Luminata Crescendo Western HRP Substrate  and detected with the Fusion Solo S system .For western blot analysis, histones were collected using a histone extraction kit . Protein concentration was determined by BCA Assay . Membranes were saturated with a solution of bovine serum albumine 5% and washed with TBS-Tween 0.2% solution after antibody incubations. The following primary antibodies were used: Rabbit anti-H3K27M , rabbit anti-H3.3 , rabbit anti-H4 , rabbit anti-H3K27me1/me2/me3 , rabbit anti-H3K27ac . All primary antibodies were diluted at Complementarily, to quantify H3K27me1/2/3 and H3K27ac marks, a multiplexed screening of histone H3 post-translational marks was also performed using the Luminex xMAP technology assay through an ActiveMotif service delivery. Briefly, 750,000 cells were harvested, centrifuged, and pellet samples were sent to ActiveMotif. Relative histone H3 concentrations in the samples were determined using the H3 Total beads. Multiplex assays were performed according to relative abundance using sample volumes normalized for histone H3 concentration. The assay plate was read on the Luminex LX200 Instrument. Samples were tested in duplicate read in a Magpix instrument and data exported as CSV files. Data sets with equivalent H3 Total signals were selected for downstream analysis. Net median fluorescent Intensity (Net MFI) associated with each PTM-specific bead was expressed as a ratio relative to Histone H3 Total signals for each well. Ratio values were averaged for each sample input amount, and percent change in the ratio relative to the reference samples was determined. T-tests at the 95% confidence interval were performed to assess PTM changes. A control lysate was also included to check the multiplex assay execution.Cells were seeded in 96-well plates (250 cells/well for SF188 and Res259 and 500 cells/well for KNS42), and the plate was placed in an Incucyte video microscope  that measured the cell confluency daily throughout the experiment.Cells were collected and incubated 30 min with Vybrant™ DyeCycle™ Violet Stain . Cell cycle was analyzed by flow cytometry using CytoFLEX , using a violet laser at 430 nm, with emission filter 450/45. Analysis was realized using Modfit software.RNA was extracted using the Nucleospin RNA kit following the manufacturer’s instructions  with on-column DNA digestion. RNA quality was evaluated using the 2100 Bioanalyzer Instrument . RIN scores above 9 were considered acceptable for RNA-seq experiments. RNA libraries were prepared using the TruSeq™ RNA Library Preparation Kit v2 and run on the Illumina NovaSeq 6000 Sequencing System  to generate 2 × 100 million paired end reads in the iGenSeq Platform of the Paris Brain Institute.https://github.com/s-andrews/FastQC; visited on 13 March 2021). Pseudo-alignment based on the human transcriptome annotation Ensembl v96 and transcript-level quantification were performed using Kallisto  [https://github.com/mikelove/tximport; visited on 13 March 2021) [https://github.com/Bioconductor/AnnotationHub; visited on 13 March 2021) [2(TPM + 2).Quality control of human pediatric glioma cell lines SF188, KNS42 and Res259 RNA-seq data was assessed using default parameters of FastQC   with “bich 2021) . Finallyhttps://github.com/saezlab/progeny; visited on 13 March 2021) on the full gene expression to quantify pathway activation for each cell line. Functional enrichment analyses were undertaken using the integrative web-based application EnrichR .Pathway RespOnsive GENes (PROGENy) is a computational method and R software package that infers pathway activity from gene expression data . This apDanio rerio) embryos were raised in a dedicated platform . Prior to injection, 2 × 106 Res259-H3.3 and Res259-H3.3K27M cells were harvested, rinsed in serum-free medium and resuspended in 30 μL of PBS. At 48 h post-fecundation, zebrafish embryos were dechorionated with pronase , anesthetized with tricaine  and injected with approximately 20 nL of either Res259-H3.3 or Res259-H3.3K27M cells in the yolk sac. Embryos were further incubated at 28 °C for 48 h in E3 medium. To quantify cells that disseminated locally in the yolk sac, embryos were fixed for 6 h in 4% paraformaldehyde (PFA) at 4 °C. Embryos were then embedded in 1% agarose columns and placed so that the yolk sac faces the lens for Selective Plane Illumination Microscopy (SPIM) imaging using a Zeiss LIGHTSHEET Z.1 with a W Plan-Apochromat 20×/1.0 detection objective . Acquisitions were realized using the Zen 2014 SP1 black edition software and 3D reconstructions were made from all z-stacks using Arivis Vision4D 3.4 . A tumor was qualified as locally disseminated when more than five cells detached from the principal mass were detected.For the local dissemination study, AB/TU zebrafish (Cells were seeded into 12-well culture plates (200 cells/well). Six hours later, cells were irradiated with fractions of 0, 1, 2 or 3 Grays per day for 3 consecutive days using a linear accelerator XSTRAHL 100 . After irradiation, cells were maintained for 10 days, then colonies were revealed by crystal violet staining. Colonies are defined to consist of at least 50 cells  and were manually counted using a colony counter pen . The Pla2. As described by Bax et al., PLOS One, 2009, low-grade  pediatric glioma Res259 cells displayed a shorter doubling time (approximately 24 h) compared to high grade  glioma SF188 and KNS42 cells . Therefore, we performed our drug sensitivity assays for 72 h according to the standards [50) were deduced from dose-response curves obtained using GraphPad Prism software.Drug sensitivity and resistance profiling was performed on human Res259, SF188 and KNS42 cell lines. The drug screening library included 80 substances consisting of a broad range of conventional chemotherapeutics and targeted agents . Each cotandards . Cell vi50 evaluation of the top 4 agents showing resistance in the K27M transfected cells was performed with the same protocol, this time with a range of 8 concentrations with a 2-fold increment between each concentration.Subsequently, precise IC2) test was used to assess the differences in the frequency of disseminated tumors compared to the total number of tumors in each group.All statistical analyses and fittings were performed with GraphPad PRISM software. Two-way ANOVA test was systematically applied to evaluate the significance of in vitro assays. Concerning experiments in zebrafish embryos, the Chi-square (χH3F3A mutated gene fused with the coding sequence for mCherry fluorescent protein in three pediatric gliomas cell lines: Res259 (WHO Grade II), SF188 and KNS42 (both WHO Grade IV). We thus obtained cells expressing an ectopic mutant histone H3.3, that we will refer to as H3.3K27M thereafter. As a control, we introduced a similar construct leading to the expression of the wild-type H3F3A gene in cells, thereafter referred to as H3.3.To investigate the impact of the H3.3K27M mutation on pediatric glioma resistance to therapies, we established stable cell lines expressing the We validated the nuclear location of our fusion proteins by fluorescence microscopy . Over 99We next investigated the expression of H3.3K27M or its wild-type counterpart in our cell lines by western blot , di- (me2) and mono- (me1) methylation by western blot. In all cell lines, the H3.3K27M mutation induced a strong decrease in the level of repressive marks, especially at the H3K27me3 level but also at the H3K27me2 one b. We conTo obtain a full insight into the molecular repercussions of the H3.3K27M mutation and of the associated epigenetic alterations, we performed a bulk RNAseq analysis to compare the transcriptome of Res259, KNS42 and SF188 cells expressing wild-type or mutant forms of the H3.3 histone. In all cell lines, we observed a wide panel of changes in gene expression patterns, with 1825, 2017, and 1275 genes whose expression was increased or decreased by more than two folds in mutant versus control cell lines in Res259, SF188, and KNS42 cells, respectively. However, only 27 genes were common to all cell lines, without enrichment in a particular signaling pathway . We coulUsing the EnrichR database, we observed that genes whose expression was changed in each cell line correspond preferentially to an enrichment in signatures associated with H3K27 trimethylation and PRC2 (Polycomb Repressor Complex), when comparing H3.3K27M vs. control cells in the three cell lines b.Collectively, these results indicate that the stable introduction of the H3.3K27M mutation in glioma cell lines is sufficient to reproduce the epigenetic changes observed in H3K27M mutated gliomas, with subsequent transcriptomic reprogramming that seems to depend on the cellular context.We then analyzed in silico the activation of distinct pathways using the PROGENy algorithm, a bio-analysis tool developed to infer the activation of specific signaling pathways. We thus observed that the expression of the K27M led to alterations of pathways involved in the regulation of cell proliferation, notably a PI3K-Akt pathway activation in Res259 and SF188 mutant lines as compared to their wild-type counterparts .We then decided to focus on the impact of the H3.3K27M mutation on glioma cell oncogenic properties. We first monitored cell growth by time-lapse microscopy and showed that Res259 and SF188 but not KNS42 H3.3K27M cells reached confluency before H3.3 control ones a. To furTo analyze the clonogenic potential of our cell lines, we then seeded them at low density in 12-well plates and quantified the proportion of cells that could form colonies. This number was much higher for Res259- and SF188-H3.3K27M cells when compared to control cells  . In contTaken together, these results suggest that H3.3K27M can modify the oncogenic properties of glioma cells, likely due to transcriptomic changes induced by the expression of the mutation.Given the diffuse nature of DIPG, we then wondered whether H3.3K27M mutation could also impact their in vivo migratory properties. We used a zebrafish xenograft model to evaluate the local dissemination of control and H3.3K27M Res259 cells . We obseFor more than 40 years, radiotherapy has remained the reference treatment for DIPG, even if these tumors eventually become refractory to this therapy. Thanks to our models, we could investigate whether the H3.3K27M mutation plays a role in this radioresistance.To address this question, we applied a fractionated protocol of ionizing radiations  to our cell models every day for 3 consecutive days. After calculating the plating efficiency of each cell line, we were able to quantify the survival rate for each dose of irradiation. No significant difference in the radiosensitivity was observed between H3.3 and H3.3K27M cells in the high-grade glioma SF188 cell line, which displayed an exacerbated basal answer to ionizing radiations, with 80% decrease in survival rate already at 3 × 2 Gy  of glioma cells. A panel of 80 pharmacological agents, including conventional chemotherapeutic drugs and targeted therapies, were tested in our glioma models. The IC50 ratio was determined for each drug in H3.3K27M versus H3.3 cell line pairs. We set the cut-off to >2.5 and <0.4, to conclude on a chemo-resistant or chemo-sensitive effect of the mutation, respectively.50 (ratio ranging from 2.5 to 24.1) and 11 drugs displaying decreased IC50 (ratio ranging from 0.36 to 0.04). This suggests a global change in DSRP in this cell model.The mutation differentially impacted the DSRP in the three cell lines a. It had50 ratios of 3.2 and 2.7 for KNS42 and Res259 cell lines, respectively); EPZ5676, that inhibits DOT1L methyltransferase ; Idelalisib, which is an inhibitor of phosphoinositide 3-kinase (PI3K) , and Alisertib, which is an Aurora A kinase inhibitor .It is noteworthy that, even if the mutation increases the chemoresistance of KNS42 cell for only six drugs, four of them exhibit a similar trend in the Res259 cells: Temsirolimus, which is an inhibitor of mTOR  and highlighted the efficiency of Panobinostat . HoweverFurthermore, we also evidenced that H3.3K27M mutation conferred resistance to alisertib and EPZ5676 , which inhibit Aurora kinase and DOT1L methyltransferase (the enzyme responsible for H3K79 methylation marks deposition), respectively.The resistance to EPZ5676 seems particularly interesting since it suggests that H3K27M-induced alterations may cross-talk with other epigenetic marks so far not investigated in DIPG, such as H3K79me. Thus, it represents a possible target to combine with other Achille’s heels of mutated DIPG cells. In this sense, future studies will be aimed at unraveling the H3K79me amount and distribution along the genome in our models in order to better understand the H3.3K27M-mediated resistance to the DOT1L inhibitor EPZ5676, and propose novel therapeutic strategies based on the highlighted mechanisms.This study was mainly carried out with in vitro models, which allowed us to combine several approaches and to obtain an integrated deeper insight into the role of H3.3K27M mutation in cell physiology and resistance to many therapies.The processes evidenced in this work, and especially the efficacy of therapies, now require to be validated with in vivo models, notably patient-derived xenografts, in order to fully meet the translational aim of the study. This will be the topic of future investigations.Although further analyses are required, these results highlight the fact that the H3.3K27M mutation can trigger mechanisms of drug/radiation resistance. Elucidation of the cross-talk between epigenetic/cellular contexts will thus be crucial to find the Achilles’ heel of tumor cells among the transcriptional alterations induced by the mutation and to devise more effective therapeutic combinations in the management of DMG.


BALF macrophage cluster FABP4+ predominant in healthy lung was transcriptionally related to STM cluster TREM2+ that governs resolution of synovitis in RA remission. Plasma concentrations of SPP1 and S100A12 (key products of macrophage clusters shared with active RA) were high in severe COVID-19 and predicted the need for Intensive Care Unit transfer, and they remained high in the post–COVID-19 stage. High plasma levels of SPP1 were unique to severe COVID-19 when compared with other causes of severe pneumonia, and IHC localized SPP1+ macrophages in the alveoli of COVID-19 lung. Investigation into SPP1 mechanisms of action revealed that it drives proinflammatory activation of CD14+ monocytes and development of PD-L1+ neutrophils, both hallmarks of severe COVID-19. In summary, COVID-19 pneumonitis appears driven by similar pathogenic myeloid cell pathways as those in RA, and their mediators such as SPP1 might be an upstream activator of the aberrant innate response in severe COVID-19 and predictive of disease trajectory including post–COVID-19 pathology.We explored the potential link between chronic inflammatory arthritis and COVID-19 pathogenic and resolving macrophage pathways and their role in COVID-19 pathogenesis. We found that bronchoalveolar lavage fluid (BALF) macrophage clusters FCN1  The severity of acute COVID-19 is attributable to genetics, immune dysregulation, abnormal blood clotting and tissue disruption, particularly implicating aberrant proinflammatory and antiviral innate immunity . RheumatFatty Acid Binding Protein 4 (FABP4) localize to the alveolar epithelial surface and recycle surfactants with type-2 alveolar epithelial cells to maintain compliance and efficient gas exchange  of healthy donors and patients with active or remission RA  in addition to hallmark proinflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α, and activated ST stromal cells. Early evidence suggested that this serum IL-6, IL-8, IL-1β, and TNF-α cytokine signature also predicts the prognosis of patients with acute COVID-19 (+ and FOLR2+ clusters) that produced abundant lipid resolvins instead of inflammatory mediators and induced a repair phenotype in ST stromal cells . Among the 9 phenotypically distinct clusters of ST macrophages (STMs) that differed in distribution between healthy donors and patients with RA, the CD48hiS100A12+ and CD48+SPP1+ clusters were expanded in RA patients with active disease  from the other BALF and STM clusters that were likely of monocytic origin (+ and FCN1+SPP1+) that dominate in severe COVID-19 share transcriptomic profiles with proinflammatory clusters  in active RA. Pearson correlation analysis between BALF FCN1+ and ST CD48hiS100A12+, and between BALF FCN1+SPP1+ and ST CD48+SPP1+, confirmed resemblance  of respective BALF/ST pairs. Independent differential gene expression analysis identified striking similarities in the pathogenic pathways of COVID-19 pneumonitis FCN1+ and active RA synovitis CD48hiS100A12+ clusters. They share 238 top marker genes , inflammation-triggering alarmins (S100A8/9/12A), B cell activation factors , promotors of IL-1β and TNF production , prothrombotic factors , and integrins mediating cell migration and adhesion . They also share receptor expression profiles — e.g., TNFR2 (TNFRSF1B), G-CSFR (CSF3R), and the complement receptor . Toc origin , 25. Theer genes  that incunctions  39). Th. Th+ andand SPP1  with ST g tissue  demonstr COVID-19, FCN+ anhi clustered tightly with the FABP4+ AM cluster in unsupervised hierarchical analysis (+ and FABP4+ unique and common cluster markers (r = 0.83 P = 2.2 × 10–16) confirmed the similarities. Differential expression analysis of their profiles identified that BALF FABP4+ and ST TREM2hi clusters share 170 markers indicating analogous homeostatic functions (C1q) that facilitates uptake of apoptotic bodies, high expression of genes of retinoic acid production  that drives Treg differentiation , (b) COVID-19 patients in their post–COVID-19 phase  at outpatient clinics (n = 41), (c) hospitalized patients with non–SARS-CoV-2 community–acquired severe acute pneumonia (n = 29), and (d) healthy donors  and S100A12  in a cross-sectional comparison of additional patient groups. These consisted of (a) hospitalized acute COVID-19 patients  clearly showed that the former had significantly higher levels of SPP1 and S100A12 (2/FiO2 ≤ 200) compared with those with lower SPP1 and S100A12 levels . St. St2/FiO S100A12 . A refin2 levels . Of prog2 levels . The tim2 levels . These d2 levels . This su2/FiO2 ratio (2/FiO2 > 200 had lower plasma GAS6 levels than those with PaO2/FiO2 ≤ 200 (2/FiO2 > 200)  . Prognos2 > 200) ; for exa2 > 200) . These d2 > 200) , 50, inc and DCs . Thus, l+FABP4 macrophages and the preferable ligand for MerTK, did not differ between any of the COVID-19 patient categories or healthy donors , and COVID-19 comorbidities  contribuneumonia . SPP1 waears old , suggestears old . GAS6 waears old ; thus, a– post–COVID-19 phase, often characterized by complex pathologies  convalescent patients reported persistence of at least 1 post–COVID-19 symptom . Increased levels of SPP1 and S100A12 persisted in all patients, although extremely high concentrations SPP1 were restricted to symptomatic patients . StratifTogether, these data suggest that the SPP1 and S100A12 myeloid cell inflammatory signature persists after resolution of SARS-CoV-2 infection and may contribute to the pathogenesis of long–COVID-19 syndrome .+ macrophages in alveoli of COVID-19 patients (n = 2), while it was rare in normal lung  and H1N1 (n = 3) pneumonia  using the immune gene panel (399 genes) with the BD_Rhapsody system after 16-hour stimulation with SPP1. We sequenced 13,580 cells and identified 14 distinct immune clusters (+ (MME+) neutrophil phenotype to a CD274+ (PD-L1+) neutrophil phenotype. The top 20 marker genes of the CD10+ and SPP1-differentiated CD274+ (PD-L1+) neutrophil clusters (+ (PD-L1+) neutrophils represent an aberrantly activated phenotype characterized by high expression of PD-L1 (CD274), which mediates suppression of adaptive immunity, high levels of proinflammatory cytokines/chemokines , c-type lectins (CLEC4E/D) that facilitate neutrophils migration, and the SPP1 receptor CD44. Similar aberrant neutrophil activation is a key pathogenic characteristic of severe COVID-19 (+ (PD-L1+) neutrophils was significantly higher in Neutrophil_2 phenotype than any other cell population , IL-1β, and chemokines  with a commensurate reduction of MHCII and antiinflammatory ENTPD1  neutrophils that suppress adaptive immunity and support inflammation.Our analysis also revealed that SPP1 induced the activation of CD14COVID-19 , 23. SPPonocytes , suggest+ and S100A12+ clusters, and their signature SPP1 and S100A12 mediators were confirmed in COVID-19 cross-sectional and longitudinal patient plasma samples. COVID-19 patients with severe respiratory distress were characterized by an aberrant raised SPP1 and S100A12 cytokine signature that could predict the urgency for ICU transfer and persisted into the post–COVID-19 phase after hospital discharge. An abundance of SPP1+ macrophages in the alveolar spaces and high SPP1 plasma levels were unique to severe COVID-19 pneumonia as compared with pneumonias induced by other pathogens. Investigation into SPP1 mechanisms of action revealed that it drives proinflammatory activation of CD14+ monocytes and development of PD-L1+ pathogenic neutrophils, both hallmarks of severe COVID-19  showed that they were the main producers of pathogenic TNF, IL-6, IL-1β, and chemokines in the synovium of RA, and this resembles the lung hypercytokine environment characterizing severe COVID-19 respiratory distress syndrome  and S100A12  and a decrease in MHCII molecules. This suggests that a sustained raised level of SPP1 may contribute to post–COVID-19 pathologies and may influence future responses against pathogens by skewing monocytes toward proinflammatory phenotype. At this stage of understanding, it remains unclear whether the plasma SPP1 and S100A12 exclusively originate from the pathogenic BALF clusters persisting in the lung of these patients. However, regardless of SPP1 and S100A12 source, their wide range of proinflammatory and fibrotic functions may be responsible for the respiratory and musculoskeletal symptoms that persist in convalescence.There is an increasing recognition of a syndrome of persistent debilitating symptoms after the COVID-19 patients become negative for SARS-CoV-2 (post–COVID-19). The symptom patterns are complex, but dyspnoea and fatigue are prominent . The patnd VLA-4 . We foun+ macrophages and homeostatic synovial lining layer TREM2+ macrophages share transcriptomic profiles. This may reflect their homeostatic functions in their respective tissues. In the synovium, the TREM2+ macrophages form a lining-layer producing and recycling lubricant synovial fluid, which facilitates joint movement  and that these were profoundly repressed in severe COVID-19. Consistent with scRNAseq data, plasma Protein S levels were higher in COVID-19 patients who maintained lung functions , suggesting a potential role in counterbalancing the severity of inflammation of SARS-CoV-2 infection.One of the aims of this study was to apply our recent discovery of inflammation-resolving macrophage pathways in RA to investigate potentially similar inflammation-resolving pathways for COVID-19. We found that tissue-resident lung alveolar FABP4movement . While iexchange . One of exchange , 60, andexchange , 62 and Limitations of the study included the younger age and fewer numbers of subjects within the healthy control group used as a comparator of some of the COVID-19 analysis. These limitations were mitigated by findings that SPP1, S100A12, and PROS1 were unaffected by age. In addition, the control group for the key mediator SPP1 in severe COVID-19 was the non–SARS-CoV-2 pneumonia group that matched COVID-19 for age, and it showed lower levels of SPP1 compared with COVID-19, supporting the link between SPP1 and COVID-19 pathogenesis.In summary, this study suggests that the pathogenesis of acute severe COVID-19 pneumonitis and RA synovitis might be driven by similar pathogenic myeloid cell clusters/pathways, producing SPP1 that persists into post-COVID-19 syndrome. Further functional studies on identification of common tissue factors driving the differentiation of these pathogenetic macrophage clusters and better understanding the functional interaction between them and the lung environment could clarify mechanisms of pneumonitis and provide evidence for potential repurposing of current antiinflammatory/antifibrotic treatments for COVID-19 , 64. ProE-MTAB-8322. COVID-19 blood and PBMC data  and ST data were acquired from EMBL-EBI:Cell-filtering involved removal of cells with < 500 expressed genes , with set-thresholds for gene-expression level, including mitochondrial genes (percent.mt) to exclude doublets and dying cells. Data normalized using Seurat’s NormalizeData function. Myeloid cells were filtered for expression of CD14, MARCO, and LYZ with the subset function. The top 2000 variable genes were identified using the FindVariableFeatures function. Sample integration of each data set followed the Seurat vignette, integrating all genes common between samples of each condition, using FindIntegrationAnchors and IntegrateData functions .UMAP based on PCA cell embeddings were generated by Seurat for each data set using RNA assay. To visualize ST data, the first 12 principal components were used (RunUMAP). These principal components determined the k-nearest neighbors for each cell during shared nearest neighbor (SNN) graph construction before clustering at a resolution of 0.5  and identifying the populations as described . In compP < 0.05 .The Seurat FindAllMarkers function identified cluster markers in individual data sets. The “test.use” function determined genes differentially expressed between clusters within each data set using MAST. As recommended, for DE comparison the nonbatch normalized counts were used. Cluster markers must be expressed by ≥ 40% cells in the cluster (“min.pct” parameter 0.4). Default values were used for all other parameters. Genes are considered significantly DE if CellTalkDB  identifiData set integration was performed as described above for 8993 common genes between ST and BALF. These “integrated” batch-corrected values were set as the default assay, and gene expression values are scaled before principal component analysis (PCA).To prevent bias in clustering, we matched BALF and ST cell numbers at 32,139 random cells. Data were rescaled , and the first 30 principal components were visualized by UMAP. Cells colored by original identity illustrated how clusters of each data set overlay.+/ST CD48hiS100A12+, BALF FCN1+SPP1+/ST CD48+SPP1+, and BALF FABP4+/ST TREM2hi clusters, respectively.A count matrix with the average expression of all common genes by each cluster was generated (AverageExpression) and hierarchical clustering  performed. Data were visualized by a dendrogram. PCA was performed on this pseudobulk expression matrix (prcomp). These analyses identified similar clusters between BALF and ST. Shared marker genes between similar clusters were identified by Venny (2.1). To visualize pseudobulk expression of overlapping marker genes, the pheatmap (1.0.12) package visualized shared genes as a heatmap using a custom script. Between–data set comparison of pseudobulk of expression marker genes  was performed by Pearson correlation  performed on the 916, the 721, and the 986 unique and common genes of BALF FCN12 saturation (>94%), or (moderate) abnormal O2 saturation (<94%); pneumonia based on imaging (chest x-ray or CT scan); and arterial oxygen partial pressure (PaO2 in mmHg) to fractional inspired oxygen (FiO2) PaO2/FiO2 (>200). Severe COVID-19 pneumonia was defined based on abnormal O2 saturation (<94%), presence of pneumonia on imaging (x-ray or CT scan), and PaO2/FiO2 (≤200), or the use of high-flow nasal cannula (HFNC), nonrebreather mask (NRB), bilevel positive airway pressure (BIPAP) or mechanical ventilation and vasopressor drugs use, creatinine clearance greater than 30, and on alanine aminotransferase (ALT) less than 5× the upper limit of normal. Concomitant comorbidities including arterial hypertension, diabetes mellitus, ischemic cardiopathy, COPD, dyslipidaemia, and cancer were recorded. The subsequent need and date of ICU admission, intubation status, and O2 therapy for each patient was recorded. Patients were treated following the internal protocol defined by the Gemelli Against COVID-19 task force as described (Reverse transcription PCR (RT-PCR) positive for SARS-CoV-2 by nasopharyngeal swab (NPS) were enrolled . Each enrolled patient provided peripheral blood samples and COVID-19 severity was classified on hospital admission and/or before antiinflammatory treatment. Mild/moderate COVID-19 pneumonia was defined based on (mild) normal Oescribed .Follow-up outpatient attendance (Fondazione Policlinico Universitario Agostino Gemelli) was offered to all discharged COVID-19 patients. Those recruited attended 68.6 ± 4.4 days (mean ± SEM) after discharge and were RT-PCR–negative for SARS-CoV-2. They were assessed by a multidisciplinary team who collected demographic and clinical data, collected blood samples, and performed physical examinations. Persistence of symptoms was recorded by a questionnaire.Patients with pneumonia, RT-PCR–negative for SARS-CoV-2 by NPS, were enrolled at the Emergency Room (Fondazione Policlinico Universitario Agostino Gemelli). Demographic and clinical parameters were recorded, and blood samples were collected. These formed a comparison pathological control group for COVID-19 pneumonia. All patients and healthy control demographics and clinical characteristics are summarized in n = 2), bacterial pneumoniae (n = 3), H1N1 pneumonia (n = 3), and normal donors (n = 3). Demographic and clinical data are summarized in www.rcpath.org). Tissue collection was performed in accordance with appropriate protocols (Postmortem lung tissue was obtained from COVID-19 pneumonia (rotocols . Lung tig/15 minutes) and plasma aliquots were stored at –80°C until analysis by ELISA for SPP1 , S100A12 , GAS6 , and PROS1 . IL-6, IL-8, TNF-α and IL-1β plasma levels were quantified by single-plex ELISA .Blood samples were centrifuged  at concentrations of 0, 50, and 200 ng/mL. After 16 hours, cells were deattached , transferred to U-bottom 96-well plates, and harvested by centrifugation at 200g for 4 minutes at 4°C. Cells from each donor/condition were labeled with unique Tags (below) using a Single-Cell Multiplexing Kit  for 20 minutes at room temperature according to manufacturer protocol.Leukocytes were collected by centrifugation from heparinized blood samples  after RBC lysis . Leukocytes were plated at 5 × 10Tag 1, ATTCAAGGGCAGCCGCGTCACGATTGGATACGACTGTTGGACCGG; Tag 2, TGGATGGGATAAGTGCGTGATGGACCGAAGGGACCTCGTGGCCGG; Tag 3, CGGCTCGTGCTGCGTCGTCTCAAGTCCAGAAACTCCGTGTATCCT; Tag 4, ATTGGGAGGCTTTCGTACCGCTGCCGCCACCAGGTGATACCCGCT; Tag 5, CTCCCTGGTGTTCAATACCCGATGTGGTGGGCAGAATGTGGCTGG; Tag 6, TTACCCGCAGGAAGACGTATACCCCTCGTGCCAGGCGACCAATGC; Tag 7, TGTCTACGTCGGACCGCAAGAAGTGAGTCAGAGGCTGCACGCTGT; Tag 8, CCCCACCAGGTTGCTTTGTCGGACGAGCCCGCACAGCGCTAGGAT; Tag 9, GTGATCCGCGCAGGCACACATACCGACTCAGATGGGTTGTCCAGG.g for 4 minutes at 4°C), after which the tagged culture variants were pooled and loaded onto the scRNAseq BD Rhapsody Cartridge using the BD Rhapsody Cartridge Reagent Kit  according to the manufacturer’s protocol. Single-cell cDNA was prepared using the BD Rhapsody cDNA Kit . This was followed by single-cell mRNA and tag-library preparation using BD Rhapsody Targeted mRNA and the Tag Amplification Kit  and primers for the BD Rhapsody Immune Response Panel . Libraries were sequenced at a depth of 1,083,775 ± 236,302 (mean ± SEM) reads per tag using Illumina NextSeq 500 (Glasgow Polyomics). Then, 1535 ± 383 cells (mean ± SEM) per tag were successfully sequenced. For analysis, the sequencing reads were processed with BD Genomics Rhapsody Analysis Pipeline CWL v.1.0 using the Seven Bridges platform. The Seurat package (3.1.5) in R was used to create an object from the RSEC_MolsPerCell.csv output file for each sample tag (CreateSeuratObject). Following the standard analysis protocol . To visualize heatmaps, the pheatmap (1.0.12) package was adapted. The normalized expression values were used to perform pseudobulk expression analysis of each sample (AverageExpression). Raw data are accessible at EMBL-EBI (E-MTAB-10430). Using Seurat (AddModuleScore), we scored the clusters of BDRhapsody COVID-19 whole-blood data set  with Dunn’s or Tukey’s correction for multiple comparisons, or 2-sided Mann-Whitney U test for 2 groups. Cytokine categories at cut-off level 108 ng/mL, 59 ng/mL, 24 ng/mL, and 15 μg/mL for SPP1, S100A12, GAS6, and PROS1, respectively, were selected based on the median levels in all COVID-19 patients. Linear correlations between cytokines and continuous parameters were performed using the Spearman’s rank test. Kaplan-Meyer analysis estimated the probability of “no need to be transferred to ICU during hospitalization” for COVID-19 patients with acute pneumonia based on the previously determined cut-off plasma values of SPP1, S100A12, GAS6, and PROS1 at the time of hospital admission. P < 0.05 was considered as statistically significant. Exact P values are provided on graphs.The scRNAseq data comparison is described in data comparison and pathway analysis. Statistical analysis of patient clinical, demographic, and plasma cytokine levels were performed using SPSS V.20.0 (SPSS) and GraphPad Prism software version 9.0.0. Categorical and quantitative variables were described as frequencies, percentage, median with the interquartile range, or mean ± SEM. Data on demographic and clinical parameters were compared between patients by Mann-Whitney The study was approved by the Committee of the Fondazione Policlinico Universitario Agostino Gemelli IRCCS — Università Cattolica del Sacro Cuore (nos. 12401/20 and 0024184/20) and University of Glasgow, MVLS College Ethical Committee (no. 2012073).MKS oversaw the project and, with SA, interpreted clinical results and, with LM, interpreted computational and experimental results. MKS, LM, SA, and CM wrote the manuscript with feedback from all authors. LM analyzed and integrated 10X Genomics scRNAseq data and interpreted all computational data. SA and BT evaluated cytokine levels in patients’ samples and performed statistical analysis with clinical data integration. LM and MKS performed SPP1 stimulation of whole blood and subsequent scRNAseq and data analysis. DS performed BD_Rhapsody sequencing library preparation and pathway analysis on scRNAseq data. LM analyzed BD_Rhapsody SPP1 data, and with DS mapped to COVID-19 data sets. TDO and MKS supervised all computational analysis in the study. BT, M. Sali, and M. Sanguinetti organized COVID-19 samples collection and handling and performed ELISA with ELLA technology. SP, AP, LP, A. Cingolani, RM, MF, MA, FL, FF, AG, A. Carfì, and EG provided clinical management of patient cohorts during acute phase hospitalization and at the postacute outpatient clinic and provided longitudinal clinical data. ES and VA provided lung tissues from cadavers and provided clinical data. AE assisted with ELISAs and preparation of tissue samples. IBM and EG assisted with running the project. EG conceived the COVID-19 translational research activity for the “Immunology Core facility” of the Fondazione Policlinico Universitario Agostino Gemelli IRCCS. Our study is a collaboration between 2 centers. The first 2 and last 2 authors contributed equally. With agreement between all authors, and reflection on each author’s contribution, we alternated the first and last authors as listed to accurately reflect the collaborative effort of the 2 centers.


The Ku703A/3A mice developed spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70+/+) littermates. The HCC tumors from the Ku703A/3A mice have increased γH2AX and 8-oxo-G staining, suggesting decreased DNA repair. Spontaneous transformed cell lines from Ku703A/3A mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70+/+ littermates. Collectively, these findings demonstrate that mutating the putative Ku70 phosphorylation sites results in defective DNA damage repair and disruption of this process drives genomic instability and accelerated development of HCC.Multiple pathways mediate the repair of DNA double-strand breaks (DSBs), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that mutating five putative phosphorylation sites on the non-homologous end joining (NHEJ) factor, Ku70, results in sustained retention of human Ku70/80 at DSB ends and attenuation of DSB repair via homologous recombination (HR). In this study, we generated a knock-in mouse, in which the three conserved putative phosphorylation sites of Ku70 were mutated to alanine to ablate potential phosphorylation (Ku70  Ku is amodeling ,4,7. Onc−/−, Ku80−/−, Ku70−/−Ku80−/− mice are viable and reproduce, but they show profound premature aging and are 40–60% the size of wild-type littermates  promotes the development of hepatocellular adenoma and hepatocellular carcinoma (HCC), which correlated with elevated chromosomal aberrations (−/− mice had accelerated HCC induction compared to wild-type and Ku70+/– littermates after treatment with the liver carcinogen diethylnitrosamine (DEN)  . The rolne (DEN) .in vitro and is required for the positional stability of DSB ends in vivo  of the conserved putative Ku70 phosphorylation sites, we aimed to test our hypothesis that initiation of HR requires dissociation of Ku70/80 from DSB ends and that blocking this process results in enhanced genomic instability and tumorigenesis. In this study, we report that blocking Ku70 phosphorylation results in increased incidence of spontaneous and accelerated induction of chemical-induced HCC. Moreover, we found that Ku703A/3A mice and MEFs are radiosensitive and that Ku703A/3A MEFs have decreased DNA end resection and increased sensitivity to the DNA cross-linking agent mitomycin C (MMC), suggesting attenuation of HR. Collectively, the results suggest that mutating the putative Ku70 phosphorylation sites results in defective DNA repair and that this drives genomic instability and development of HCC.A secondary function that Ku performs at DSBs is a general role of protecting DNA ends from non-specific processing, which if left unchecked could lead to genomic instability ,6,23. Ku in vivo ,25. Ku b in vivo . As DNA  in vivo . This is in vivo ,28. It h in vivo . Previou in vivo . This st+/+ and Ku703A/3A mice were cultured in Hyclone α-MEM (GE Life Sciences) supplemented with 10% fetal bovine serum (Corning) and 1x penicillin/streptomycin (GE Life Sciences) and initially grown in an atmosphere of 3% O2 and 10% CO2 at 37°C in order to drive spontaneous transformation. Following transformation, the primary MEFs were grown in the same media in an atmosphere of 5% CO2. Ku70−/− MEFs (2 at 37°C. Ku70−/− MEFs complemented with mouse wild-type Ku70 and Ku70 3A were maintained in the same media but supplemented with 10 μg/ml hygromycin (Corning).Primary MEFs isolated from E13.5 embryos from Ku70−/− MEFs  were gro137Cs irradiator (J.L. Shepherd and Associates) at the doses denoted in the figures.Cells were irradiated with γ-rays generated by a Mark 1 Immunoblotting was performed as previously described . The folBamHI and XhoI following standard protocols. PCR-directed mutagenesis using complimentary oligonucleotides was then used to generate mouse Ku70 3A. The following primers were used for the mutagenesis: mKu70-307A: 5′-GACTTTTAATGTAAACGCCGGCAGTCTACTCC-3′ and mKu70-314A/316A: 5′-CAGTCTACTCCTGCCTGCTGACGCCAAGCGGTCTCTGAC-3′ with the mKu70-307A primer set used first and then the mKu70-314A/316A primer set. To make Ku70−/− MEFs that stably express wild-type and Ku70 3A, 4 μg of linearized plasmid containing wild-type or Ku70 3A cDNA was transfected using Amaxa nucleofector solution V (Lonza) and program T-020. Cells stably expressing mKu70 were selected using 800 μg/ml hygromycin.Mouse Ku70 cDNA was PCR amplified using the primers mKu70-Bam-ATG: 5′-GGCGGATCCATGTCAGAGTGGGAGTCCTAC-3′ and mKu70-Stop-Xho: 5′-GGCCTCGAGTCAGTTCTTCTCCAAGTGTCTG-3′ and then subcloned into the pCDNA3.1-YFP-F2 mammalian expression vector using Live cell imaging combined with laser micro-irradiation was performed as previously described ,7. FluorIR-induced RPA/Rad51 kinetics were determined as previously outlined . RPA andIR-induced 53BP1 kinetics were determined as previously outlined with modifications . Cells wAll mice were approved by and handled in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at UT Southwestern Medical center (UTSW). All mice were bred and maintained in a specific pathogen free SPF barrier vivarium at UTSW. All experiments were conducted with age and sex matched mice and treatment groups were allocated randomly.GCCGGCAGTCTACTCCTGCCTGCTGACGCCAAGCGGTCTCTGGTAGGTGGCTAACCTTTCCTACCGAATCTTGTTTAAGA-3′ (the mutation sites are underlined). After microinjection, the zygotes were implanted into ICR recipients, which carried them to term. Seven chimeras  were obtained and then backcrossed twice with wild-type C57BL/6J mice (Jackson Laboratory). For sequencing and PCR genotyping, DNA was isolated from either the mouse tail or ear using a previously described protocol  were induced to superovulate by injecting pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) and then mated with C57BL/6N males to collect the zygotes for microinjection. A mixtures of Cas9-mRNA (Sigma) (50 ng/μl), synthesized gRNA (Sigma) (25 ng/μl) and template single-strand oligodeoxynucleotide (ssODN) (Sigma) (25 ng/μl) were injected into pronuclear of one-cell zygotes. The Ku70 gRNA sequence utilized is 5′-ACACCAAGCGGTCTCTGGTAGG-3′ and the template ssODN (donor oligo) sequence is 5′-GAACCAGTGAAAACCAAGACAAGGACTTTTAATGTAAACprotocol . For seq3A/3A, Ku703A/+ and Ku70+/+ littermates were maintained and monitored for up to 28 months. Necropsy was performed at various time points and the mice were examined for gross tumors. All tissues with tumors were collected and fixed with 4% neutralized PFA for 24 hours. Clinical histopathological analysis of H&E-stained sections were performed blindly.Ku703A/3A and Ku70+/+ littermates were injected with DEN (Sigma) (25 mg/kg i.p.) intraperitoneally (i.p.) at day 14 postpartum. All mice were monitored weekly for weight and activity and sacked at different intervals  as appropriate to test for liver disease. Nine months following DEN injection, all surviving mice from all genotypes were euthanized with CO2 and their liver tissue was collected and fixed. DEN-induced liver tumor numbers were determined visually, and size determined using a Vernier caliper.Male C57BL/6J Ku70The fixed tissues samples were processed for paraffin sectioning and stained with hematoxylin-eosin (H&E) according to standard protocol by the Histo-Pathology Core and Tissue Management Shared Resource (TMSR) at UTSW. IHC staining was performed using the following antibodies, Phospho-Histone H2A.X (Ser139) (20E3) Rabbit mAb , Ki-67 (D3B5) Rabbit mAb , and DNA/RNA damage antibody (8-oxo-G) [15A3] (Abcam) by the TMSR at UTSW.3A/3A mice and Ku70+/+ littermates were irradiated with 9 Gy total body irradiation (TBI) utilizing the self-contained X-ray irradiation system . Their weight and activity were monitored daily until 30 days post-irradiation. The mice were sacked when they lost more than 20% of their initial body weight; their Body Conditioning Score (BCS) fell below 2%, or at the date of experimental termination.Healthy 8-week-old Ku70+/+ or Ku703A/3A (clones #51 and #57) were treated with increasing doses of DNA damaging agents  and survival assay were performed as previously outlined (+/+ or Ku703A/3A (clones #51 and #57) were pre-treated with 1 μM of the DNA-PKcs inhibitor NU7441  for 2 h before irradiation in the experiment shown in Figure For clonogenic survival assays, DC-1 Ku70 null) and DC-1 complemented with wild-type mouse Ku70 or Ku70 3A protein or spontaneously transformed MEFs from Ku700 null anoutlined . FurtherFor chromosomal abnormalities, spontaneously transformed primary MEFs (passages 8–10) were incubated with 200 ng/ml MMC (Sigma) for 20 h followed by addition of 0.1 μg/ml Demecolcine Solution (Sigma) in the last 4 h. Samples were processed and chromosomal abnormalities were scored as previously described .Total immune cells from the spleen were isolated following previously reported methods .For cell surface staining, lymphocytes isolated from spleens were mixed with anti-mouse CD16/32 mAbs on ice for 5 min to block Fc receptors. Cells were mixed with Ab cocktail containing mAbs against specific cell-surface markers and incubated on ice for 30 min in the dark. The following antibodies were used , Panel 1  and Panel 2 . The stained samples were transferred to 5-ml flow tubes and 200 μl of staining buffer was added to each sample. The samples were run on a FACS Calibur (BD) and data analyzed using FlowJo software by the Flow Cytometry Core at UTSW.−/− cell line DC-1  using CRISPR/Cas9  developed normally , T-cell sub-populations  and Natural Killer (NK) cells or myeloid cell populations  between Ku703A/3A and Ku70+/+ littermates J recombination  of male mice developing HCC   in Ku703A/+ mice compared to Ku70+/+ mice  staining with the proliferation marker Ki67, DSB marker γH2AX, and free radical damage/reactive oxygen species (ROS) marker 8-oxoguanine (8-oxo-G). IHC analysis of the HCC tumors from Ku703A/3A revealed strong Ki67, γH2AX and 8-oxo-G staining, suggesting high levels of proliferation and accumulated DNA damage in the HCC tumors  promotes accelerated HCC development in Ku703A/3A mice  of DEN and were examined at 4-, 6-, and 9-months post-injection for liver tumors. A minimum of 6 (and up to 11) animals from each genotype were analyzed. There was no evidence of liver neoplasia in the Ku70+/+ mice at 4 months, whereas 6 out of 7 Ku703A/3A mice had at least 1 liver tumor at this time point (average of 1.14 ± 0.26 tumors/mouse)  than those found in Ku70+/+ mice (0.91 ± 0.34 mm3)  compared to Ku70+/+ mice (7.33 ± 2.07) with no difference in average tumor size between the two cohorts observed  higher at 4 months, 5.6 times (95% CI 2.65–12.01) higher at 6 months, and 1.7 (95% CI 0.77–3.86) times higher at 9 months compared to Ku70+/+ mice  Figure  and B. Fe Figure  and B. A) Figure . Expectee Figure . Control3A/3A mice have accumulated DNA damage, suggesting defective DNA repair in this cohort of mice. To test if the Ku703A/3A mice have defective DSB repair compared to their Ku70+/+ littermates, total body irradiation (TBI) experiments were performed. Treatment with 9 Gy of X-rays revealed that Ku703A/3A mice are significantly radiosensitive compared to Ku70+/+ mice  of Ku70+/+ mice had perished from the radiation exposure. No statistical difference between male and female Ku703A/3A mice in radioresponse was observed  symptoms, as intestinal H&E staining showed significant intestinal crypt and villi destruction in the Ku703A/3A mice compared to Ku70+/+  and immunoblotting showed similar protein expression of Ku70 and Ku80 in these MEFs . MMC-induced ICLs are effectively resolved/repaired via the Fanconi anemia (FA) pathway in conjunction with HR , suggesting there is a modest decrease in NHEJ in the Ku703A/3A MEFs. Moreover, IR survival assays in the presence of the DNA-PKcs inhibitor NU7441 showed that Ku703A/3A MEFs are more sensitive than Ku70+/+ MEFs, implicating that NHEJ is not fully defective in Ku703A/3A MEFs and that the increased radiosensitivity is due to decreased HR and NHEJ  mice, as ablating the putative Ku70 phosphorylation sites does not drive gross defects in immune cell production or premature aging, suggesting that V(D)J recombination and telomere maintenance are not affected, respectively  gene in and near the Ku70 phosphorylation cluster, including at the conserved phosphorylation sites S314  and T316  (XRCC6 (Ku70), XRCC5 (Ku80) and PRKDC (DNA-PKcs), to the sequencing studies examining mutations of DDR proteins in carcinogenesis (The role of Ku in the pathogenesis and development of human HCC remains unclear. One report found that Ku80 is frequently downregulated in human HCC . Howevery to HCC . Sequenc cancer) . Mutatio cancer) . Collect cancer) . As therogenesis ,49.+/+ mice, the DNA damage is rapidly repaired and genomic stability is maintained in the liver. However, in Ku703A/3A mice, mutating the putative Ku70 phosphorylation sites attenuates the repair of DSBs that are directly generated or those created due to the processing of DNA adducts, SSBs, or ICLs. This is supported by our data showing that the HCC tumors identified in Ku703A/3A mice have increased unrepaired DSBs as assessed by γH2AX staining and Ku703A/3A MEFs are sensitive to IR and MMC compared to MEFs from wild-type littermates. We postulate that the decrease in DSB repair capacity observed in Ku703A/3A mice and MEFs is due to a marked reduction in HR and a slight decrease in NHEJ. The decrease in HR is due to sustained retention of the Ku70 3A protein at DSBs, which attenuates DNA end resection. Recently, we published a study , IR , and MMC (ICLs and ROS-induced DNA damage), which supports the notion that Ku703A/3A mice may have issues repairing multiple types of DNA damage . The liver is constantly under attack from multiple agents, such as carcinogens (aflatoxins), metabolites , and reactive oxygen species (ROS) from inflammation due to obesity. We postulate that these agents generate various DNA damage including DNA adducts, SSBs, DSBs, and ICLs. In Ku70 a study  that repA damage . One of nduction . Acetaldnduction . In micenduction . Ku703A/e damage . The Ku e damage . Collectanthony.davis@utsouthwestern.edu) for reagents.All study materials will be made available to other researchers. Please contact Anthony J. Davis (gkab743_Supplemental_FileClick here for additional data file.


The continuous evolution of the H7N9 avian influenza virus suggests a potential outbreak of an H7N9 pandemic. Therefore, to prevent a potential epidemic of the H7N9 influenza virus, it is necessary to develop an effective crossprotective influenza vaccine. In this study, we developed H7N9 virus-like particles (VLPs) containing HA, NA, and M1 proteins derived from H7N9/16876 virus and a helper antigen HMN based on influenza conserved epitopes using a baculovirus expression vector system (BEVS). The results showed that the influenza VLP vaccine induced a strong HI antibody response and provided effective protection comparable with the effects of commercial inactivated H7N9 vaccines against homologous H7N9 virus challenge in chickens. Meanwhile, the H7N9 VLP vaccine induced robust crossreactive HI and neutralizing antibody titers against antigenically divergent H7N9 viruses isolated in wave 5 and conferred on chickens complete clinical protection against heterologous H7N9 virus challenge, significantly inhibiting virus shedding in chickens. Importantly, supplemented vaccination with HMN antigen can enhance Th1 immune responses; virus shedding was completely abolished in the vaccinated chickens. Our study also demonstrated that viral receptor-binding avidity should be taken into consideration in evaluating an H7N9 candidate vaccine. These studies suggested that supplementing influenza VLP vaccine with recombinant epitope antigen will be a promising strategy for the development of broad-spectrum influenza vaccines. More importantly, some novel biological features of the H7N9 virus, such as immune escape mutations and antigenic drift were discovered in H7N9 variants  was used as a vaccine supplement in the present study. The result demonstrated that the influenza VLP subunit vaccine induced robust HI and neutralizing antibody titers to crossprotect against challenge with a lethal homologous and heterologous H7N9 virus. The influenza VLP vaccine supplement with HMN or Poly(I:C) enhanced a Th1-biased influenza-specific immune response in chickens, which was significantly inhibited virus shedding.The main goal of this study was to develop an influenza VLP subunit vaccine and an effective supplement vaccination strategy to provide crossprotection against an influenza virus challenge. A recombinant protein (HMN) consisting of three tandem conserved epitopes: two repeats of HAAll experiments involved in the live H7N9 avian influenza viruses (AIVs) were performed in a biosafety level 3 laboratory facility at South China Agricultural University (SCAU) (CNAS BL0011) in accordance with protocols. All animals involved in the experiments were reviewed and approved by the Institution Animal Care and Use Committee at SCAU and treated in accordance with the guidelines (2017A002).Spodoptera frugiperda 9 (Sf9) and BTI-TN-5B1-4 (High Five™) insect cells were used in this study. Sf9 cells  were maintained in Sf-900 II serum-free medium  and used for the production of recombinant baculovirus. High Five™ cells  were maintained as a suspension in HF-SFM  in shaker flasks at a speed of 100–120 rpm and used for the production of recombinant proteins. Both insect cell lines were cultured at 27°C. Madin-Darby canine kidney (MDCK) cells were maintained at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal bovine serum .g centrifugation for 5 min. The clarified fluid was then ultracentrifuged at 30,000×g for 1 h, and the virus solution was further purified using a 20%–30%–45%–60% discontinuous sucrose gradient. The 50% egg infectious dose (EID50) and the 50% egg lethal dose (ELD50) were calculated using the Reed-Muench method (HPAI H7N9 viruses A/Chicken/Guangdong/16876/2016 (H7N9-16876) , A/ChickTo generate the VLP, the hemagglutinin (HA), neuraminidase (NA), and matrix protein (M1) genes derived from A/Chicken/Guangdong/16876/2016(H7N9) were biochemically synthesized by BGI . Genes of HA, NA, and M1 were codon optimized for a high level of expression in High Five cells, then a 6xHis epitope tag was simultaneously fused to the C-terminal end of the optimized gene.76-130, 4M2e, 8NP55-69, and a flexible linker sequence (3xG4S) was designed and named HMN (55-69 sequence was linked by a linker sequence (GGSS), and the 6xHis tag epitope was linked to the 3′ ends of the HMN sequence by a GGSS linker. HMN gene was codon optimized for a high level of expression in the High Five cells and synthesized by BGI.A recombinant chimeric protein containing honeybee melittin signal peptide, tandem repeat of 2HAamed HMN Table 1.Escherichia coli DH10Bac to make recombinant bacmid baculovirus DNA, purified recombinant bacmid DNAs were transfected into sf9 insect cells using Cellfectin™ II reagent (Invitrogen) to obtain the recombinant baculovirus (rBV) in the culture supernatant. Following the manufacturer’s instructions, the recombinant baculoviruses were then amplified by infecting sf9 insect cells. All preparations of rBV were plaque purified and titrated using a rapid titration kit .These four optimized genes were cloned into the pACEBac1 vector plasmid , respectively. The recombinant plasmids were transformed into g for 30 min at 4°C to remove debris. The VLPs in the supernatants were purified by ultracentrifugation at 30,000×g for 60 min at 4°C. The sedimented particles were resuspended in phosphate-buffered saline  at 4°C overnight and further purified through a 20%–30%–45%–60% discontinuous sucrose gradient at 100,000×g for 1 h at 4°C  in shaker flasks at 27°C. For the production of VLP containing the H7N9 HA, NA, and M1 proteins, High Five cells were coinfected with rBVs expressing HA, NA, and M1, respectively, at a multiplicity of infection (MOI) of 2:1:2. After 3 days postinfection, cell culture supernatants were harvested by centrifugation at 2,000×h at 4°C . The fung for 3 min at 4°C) to remove cell debris. The target proteins were purified using Ni-chromatography and used for further studies. The concentration of the purified VLPs and HMN was quantified using the Pierce BCA Protein Assay Kit .For the production of HMN proteins, High Five cells were infected with rBV expressing HMN protein in shaker flasks at an MOI of 1. After 3 days postinfection, the infected High Five cells were harvested and disrupted by ultrasonication for 30 min to prepare cell lysates under the condition of maintaining the temperature at 0°C–4°C. The sonicated cell lysates were cleared by low-speed centrifugation  was performed to detect the expression of VLPs and HMN protein in infected sf9 insect cells. Briefly, sf9 insect cells were infected with recombinant baculovirus expressing H7N9 proteins or HMN, respectively. After incubation for 48 h, the cells were fixed with 80% precooled acetone at −20°C for 15 min and incubated with the primary chicken antiserum against H7N9 AIVs or anti-His-tag mouse monoclonal antibody at a dilution of 1:200, and then with the secondary fluorescein isothiocyanate (FITC)-conjugated goat anti-chicken IgG antibody  or rabbit anti-mouse IgG antibody . Fluorescent images were examined under an inverted fluorescence microscope .The H7N9 VLPs and HMN proteins were analyzed using SDS-PAGE and Western blot. Briefly, the protein samples were mixed with 5x SDS-PAGE loading buffer  and boiled for 10 min, then separated by 10% Tris-Glycine gels, and stained using Coomassie Brilliant Blue  for SDS-PAGE analysis. The protein bands were also transferred to nitrocellulose membranes  for Western blot analysis. The membranes were blocked with 5% (W/V) skim milk in PBST [PBS containing 0.05% (v/v) Tween 20] overnight at 4°C. Membranes were subsequently incubated with an anti-His-tag mouse monoclonal antibody  for 1 h at room temperature. The blots were then washed five times with PBST and incubated with a horseradish-peroxidase-conjugated goat anti-mouse IgG antibody  for 1 h at room temperature. Finally, the proteins were visualized by chemiluminescence (LI-COR Odyssey).Sucrose gradient-purified VLP samples were adsorbed onto a carbon parlodion-coated copper grid for 2 min. Excess VLP suspension was removed by blotting with filter paper, and the grid was immediately stained with 1% phosphotungstic acid for 10 min. Excess stain was removed by filter paper, and the samples were examined using a transmission electron microscope .N = 10) of chickens were subcutaneously immunized once with 30 μg of  VLPs in combination with EOLANE 150 . The commercial avian influenza trivalent inactivated vaccine (H7+H5) was set as comparison control, and one group of chickens was inoculated with PBS as a negative control. Three weeks after immunization, chickens were intranasally challenged with 2 × 106.0 ELD50 (0.2 ml) HPAI H7N9-16876.Three-week-old SPF chickens were purchased from the Experimental Animal Center . They were maintained according to the South China Agricultural University’s guidelines for the care and use of laboratory animals and used to determine the immunogenicity and efficacy of the H7N9 VLPs. The commercial avian influenza trivalent inactivated vaccine  was provided from South China Biological Medicine Co., Ltd. . For a homologous protection study, a group (N = 13 each group) of chickens were subcutaneously immunized once with 30 μg of VLP with ISA 201 VG, ISA 201 VG supplemented with 30 μg of HMN, and ISA 201 VG supplemented with 30 μg of Poly(I:C) ; one group (N = 13) of chickens was immunized intramuscularly once with 30 μg of VLP with ISA 71VG , and one group of chickens was inoculated PBS as a negative control. Nineteen days after immunization, the peripheral blood and spleen of chickens (N = 3) in each group were obtained for the determination of cytokine levels. Three weeks after immunization, other chickens (N = 10) of each test group were inoculated intranasally with 106.0 EID50 of H7N9-E157 virus in a 200-μl volume. Chickens were monitored for clinical signs and mortality for 14 days postchallenge (PC). All surviving chickens were killed humanely at the end of monitoring experiments.For a crossprotection study, groups . After incubation for 48 h at 37°C, the allantoic fluids were tested for hemagglutination activity. A virus isolation positive swab means one or more of the inoculated egg allantoic fluids reciprocal to the hemagglutination titers was higher than 4.To determine virus positivity or shedding from individual chickens, the oropharyngeal and cloacal swab samples were collected at 5 days postchallenge in the homologous protection study. The oropharyngeal and cloacal swab samples were collected at 3, 5, 7, and 9 days postchallenge in the crossprotection study. The swab samples were resuspended in 1 ml of PBS supplemented with 2,000 mg/ml streptomycin and 2,000 IU/ml penicillin. The suspensions were centrifuged at 3,000×To determine the immunogenicity of the vaccines, serum antibody levels were titrated by hemagglutination inhibition (HI) assay or neutralization assay. Hemagglutination inhibition (HI) assay was performed using standard methods . Brieflyl-glutamine  at a final concentration of 1 × 106 cells/ml. Cells were stimulated with 20 μg of inactivated H7N9-E157 virus or H7N9 VLPs and incubated for 8 h at 37°C. Cells were then harvested for RNA extraction. Cytokine expression levels of cells were evaluated using qRT-PCR.Peripheral blood mononuclear cells (PBMCs) and splenocytes were prepared for cytokine assays. PBMCs were isolated from peripheral blood using Ficoll-Hypaque density sedimentation . Splenocytes were obtained from the spleens of chickens by density gradient centrifugation using Lymphoprep  according to the manufacturer’s instructions. After contaminating red blood cells (RBC) present in the isolated cells lysed using RBC lysis buffer , single cells were collected. PBMCs and splenocyte single-cell suspensions were cultured in complete Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% FBS and 1% penicillin-streptomycin/Total mRNA was extracted using total RNA extraction kits ; 500 ng of total mRNA was converted into cDNA using HiScript Reverse Transcriptase  according to the manufacturer’s instructions. mRNA expressions were examined using quantitative real-time PCR (qRT-PCR) with ChamQ Universal SYBR qPCR master mix  using a Bio-Rad CFX Applied System PCR instrument . Sequences of primers used for qRT-PCR are shown in *p < 0.05, **p < 0.01, ***p < 0.001, or ****p < 0.0001.Experimental data are presented as mean ± SD of the mean. GraphPad Prism 7 software was used for data analysis. The results of serum antibody titers and cytokine level were evaluated using one-way ANOVA and Tukey’s multiple-comparison test. Significant differences are denoted by an asterisk as follows: 13. The size and morphology of H7N9 VLPs were examined by transmission electron microscopy  expressing HA, NA, and M1. Based on optimization results, the H7N9 VLPs in the study were produced using High Five cells coinfected with rBVs expressing HA, NA, and M1 at an MOI of 2:1:2. The expression of H7N9 proteins was observed with indirect IFA with chicken antiserum against H7N9 AIVs in sf9 cells 48 h after coinfection with HA, NA, and M1 rBVs  of A/Chicken/Guangdong/16876/2016 (H7N9) virus 3 weeks after immunization. The survival rates and morbidity of chickens in each group were monitored for 2 weeks after the challenge. All chickens in the H7N9 VLPs and H7N9 commercial vaccine group survived the infection. In contrast, all chickens in the control group died of infection 2 days postchallenge ] from the fifth wave increases the viral receptor-binding avidity to RBC, leading to decreasing HI titers against viruses containing HA Q226 and resulting in a biased antigenic evaluation based on HI assay . In thisin vitro. The level of the cytokines IFN-γ, IL-4, and IL-17, associated with Th1-type, Th2-type, and Th17-type immune responses, respectively, were determined to evaluate the immune types induced by H7N9 VLP vaccine candidates. After virus stimulation in vitro, mRNA expression levels of IFN-γ and IL-4 were significantly higher in the PBMCs of chickens that received ISA 71 VG-adjuvanted vaccine than levels of IFN-γ and IL-4 in the PBMCs of chickens that received ISA 201 VG-containing adjuvanted vaccine. The mRNA levels of IFN-γ were significantly higher in the PBMCs of VLPs in combination with ISA 71 VG-vaccinated chickens than that of the IL-4, indicating that Th2-biased immune responses were induced by ISA 71 VG-adjuvanted vaccine  or HMN, respectively  of the H7N9-E157 virus 3 weeks after immunization. All chickens in the vaccine groups of VLP+ISA 201, VLP+ISA 201+HMN, VLP+ISA 201+Poly(I:C), and VLP+ISA 71 survived the infection. In contrast, all chickens in the control group died of infection 3 days PC , and VLP+ISA 71 vaccines, respectively; the control group was treated with PBS. All chickens were intranasally challenged with 10 days PC Figure 8via the oropharynx and cloaca were analyzed to determine the virus replication at 3, 5, 7, and 9 days PC  were performed in a biosafety level 3 laboratory facilities at South China Agricultural University (SCAU) (CNAS BL0011) in accordance with protocols. All animals involved in the experiments were reviewed and approved by the Institutional Animal Care and Use Committee at SCAU and treated in accordance with the guidelines (2017A002).HF, ML, and DK designed the research. DK, TC, XH, SL, and YG performed the experiments. DK, TC, and CJ analyzed the data. DK, ML, and HF participated in writing the paper. All authors reviewed the manuscript.This work was supported by the Innovation Leading Team Program of Guangzhou City (202009020009) and the Key Research and Development Program of Guangdong Province (2019B020218004).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In vitro, A438079 reduced high glucose (HG) induced cell damage in H9c2 cells and primary rat cardiomyocytes. Furthermore, HG/streptozotocin (STZ)-induced P2X7R activation mediated downstream protein kinase C-β (PKCβ) and extracellular regulated protein kinases (ERK) activation. This study provided evidence that P2X7R plays an important role in the pathogenesis of STZ-induced diabetic cardiac damage and remodeling through the PKCβ/ERK axis and suggested that P2X7R might be a potential target in the treatment of diabetic cardiomyopathy.Diabetic cardiomyopathy (DCM) is a complication of diabetes mellitus which result in cardiac remodeling and subsequent heart failure. However, the role of P2X7 receptor (P2X7R) in DCM has yet to be elucidated. The principal objective of this study was to investigate whether P2X7R participates in the pathogenesis of DCM. In this study, the C57BL/6 diabetic mouse model was treated with a P2X7R inhibitor (A438079). Cardiac dysfunction and remodeling were attenuated by the intraperitoneal injection of A438079 or P2X7R deficiency.  Many patients are diagnosed with diabetes worldwide, and the complications caused by diabetes are also diverse, such as diabetic nephropathy, diabetic retinopathy, and diabetic heart diseases. Among these complications, diabetic cardiomyopathy (DCM) is a serious complication that results in a poor prognosis for patients with diabetes . In indi+, Na+, and Ca2+ plasma permeation (predominantly Ca2+) , which is composed of 595 amino acids, is a non-selective cationic gated channel that assembles into a trimeric complex and belongs to the P2X family . After bly Ca2+) . The P2Xrophages . Accordirophages . Simultarophages . In diabrophages . P2X7Ri rophages . Howeverin vivo and in vitro. In addition, we further described the crucial role of P2X7R in knockout mice with diabetes.The aim of the present study was to elucidate the essential role of the P2X7R in regulating DCM and its underlying mechanisms. The P2X7R-selective inhibitor A438079 was used in a streptozotocin (STZ)-induced type I diabetes mouse model, cultured H9c2 cells, and primary rat cardiomyocytes. The expression of P2X7R was significantly upregulated in the STZ-induced diabetic mouse model and high glucose (HG)-treated cell model. Moreover, P2X7R inhibition dramatically relieved cardiac damage by ameliorating fibrosis, apoptosis, and cardiomyocyte hypertrophy in STZ- or HG-induced models –/– mice on a C57BL/6 background were provided by GemPharmatech Co., Ltd. . Non-knockout male C57BL/6 mice were obtained from the Animal Center of Wenzhou Medical University. Animals were housed on a 12:12 h light–dark cycle at a constant room temperature and fed a standard rodent diet. The animals were acclimated to the laboratory for at least 2 weeks before initiating the studies. All operations were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal care and experimental protocols were approved by the Committee on Animal Care of Wenzhou Medical University.Male C57BL/6 wild-type mice and male P2X7R–/– mice were randomly divided into four groups: (i) non-treated C57BL/6 control mice that received buffered saline ; (ii) non-treated P2X7R–/–control mice that received buffered saline ; (iii) STZ-induced diabetic mice ; and (iv) P2X7R–/– mice that received an i.p. injection of STZ . Non-knockout male C57BL/6 mice were randomly divided into four groups: (I) non-treated C57BL/6 control mice that received buffered saline ; (II) STZ-induced diabetic mice without treatment ; (III) STZ-induced diabetic mice that received an i.p. injection of 10 mg/kg A438079 every second day for 16 weeks ; and (IV) STZ-induced diabetic mice that received an i.p. injection of 20 mg/kg A438079 every second day for 16 weeks . All animals were sacrificed by administering sodium pentobarbital anesthesia. The hearts were collected and weighed. In addition, heart tissues were placed in 4% paraformaldehyde for the pathological analysis and/or snap-frozen in liquid nitrogen for gene and protein expression analyses.Eight- to twelve-week-old mice weighing 23–25 g were used to develop the diabetes mellitus model. Diabetes mellitus was induced by a single intraperitoneal (i.p.) injection of STZ  at a dose of 50 mg/kg formulated in 100 mM citrate buffer (pH 4.5) for 5 consecutive days. After 1 week, blood samples were collected. Fasting blood glucose levels were measured using a glucometer after a 12 h fast. Mice with fasting blood glucose levels >12 mmol/L were considered diabetic and chosen for further experiments. C57BL/6 wild-type mice and P2X7RSystolic and diastolic cardiac function were determined non-invasively using transthoracic echocardiography in mice 2 h before sacrifice. Diastolic function was assessed by performing pulsed-wave Doppler imaging of the transmitral filling pattern. The ejection fraction (EF) was calculated from the left ventricle end-diastolic volume (LVEDV) and end-systolic volume (LVESV) using the following equation: (LVEDV−LVESV)/LVEDV× 100%. Fractional shortening (FS) was calculated using the following equation: FS = [(LVIDd−LVIDs)/LVIDd]×100%. The Tei index was determined based on the Doppler recordings of the left ventricular isovolumetric relaxation time (IRT), isovolumetric contraction time (ICT), and ejection time (ET):Tei=(IRT+ICT)/ET.Newborn (2–3 days old) SD rats were obtained from the Animal Center of Wenzhou Medical University. The incision on the sternal left margin was disinfected with 75% alcohol. The hearts were removed from the chest, washed twice with cold PBS, and gently dissected. The heart fragments were digested with 0.08% trypsin for 8 min at 37°C with magnetic stirring. This step was repeated approximately 16 times until the tissue organization was no longer visible. The supernatant was collected, and the digestion was terminated by an incubation with DMEM containing 10% FBS. All the collected liquid was centrifuged at 1000 rpm for 10 min. The cells were incubated for 1 h at 37°C after suspension. The non-adherent cells were primary cardiomyocytes, and the adhesive cells were fibroblasts, which were all collected and cultivated for the experiment.D-glucose, 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37°C in a 5% CO2 incubator . Experiments were performed when the cell density is about 70–80%. In the HG-treated group, H9c2 cells or primary cardiomyocytes were incubated with DMEM containing 33 mmol/L glucose. A438079, LY317615, and PD98095 were dissolved in DMSO and added to the cells for 1 h. The final concentration of the three inhibitors was 10 μM. Afterward, the cells were exposed to HG for 24 h.The rat myocardium-derived cell line H9c2 was obtained from the Shanghai Institute of Biochemistry and Cell Biology . H9c2 cells or primary cardiomyocytes were maintained in DMEM  containing 5.5 mmol/L TM (TliRnaseH Plus) was obtained from Takara . The primary anti-P2X7R antibody (#APR-004) was obtained from Alomone Labs (Israel). Anti-collagen 1 , anti-matrix metalloproteinase-9 , and anti-heavy chain cardiac myosin (ab185967) antibodies were obtained from Abcam . Moreover, antibodies against TGFβ (#3711), GAPDH (#5174), Caspase-3 (#9662), Bcl2 (#2870), phospho-PKCβII (Thr638/641) (#9375), Bax (#5023), and Phospho-p44/42 MAPK (ERK1/2)  were obtained from Cell Signaling Technology .The P2X7R inhibitor (A438079), PKC inhibitor (LY317615), and ERK-specific inhibitor (PD98095) were purchased from Selleck . A RevertAid First Strand cDNA Synthesis Kit was obtained from Thermo Fisher Scientific . SYBR Premix Ex TaqProtein isolation and western blot analyses were performed using methods described in the literature . ProteinTotal RNA was extracted from H9c2 cells or heart tissues (20–50 mg) using TRIzol reagent according to the manufacturer’s protocol (Invitrogen Life Technologies). One microgram of total RNA from each sample was used to generate cDNAs using the RevertAid First Strand cDNA Synthesis Kit . The resulting cDNA templates were amplified using SYBR in a real-time polymerase chain reaction with primers from Sangon Biotech  . PCR wasParaffin tissue sections with a thickness of 5 μm were used for terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of apoptotic cells using a One-Step TUNEL Apoptosis Assay Kit from Beyotime  according to the manufacturer’s instructions. TUNEL-positive cells were imaged under a fluorescence microscope .Excised heart tissue specimens were fixed with 4% paraformaldehyde, processed in a graded series of alcohol and xylene solutions and then embedded in paraffin. Paraffin blocks were sliced into sections at a thickness of 5 μm. After rehydration, the sections were stained with hematoxylin and eosin (H&E). Images of each section were captured using a light microscope  to evaluate the histopathological damage. Paraffin sections (5 μm) were stained with 0.1% Sirius Red and Masson’s trichrome (Sigma) to evaluate the level of collagen deposition and fibrosis, respectively. H9c2 cells were pretreated with HG (33 mM) for 24 h in the presence or absence of A438079. Then, the cells were stained with rhodamine  according to the manufacturer’s instructions. The stained sections or cells were then viewed under a Nikon fluorescence microscope .H9c2 cells were pretreated with HG (33 mM) for 24 h before the level of apoptosis was measures using flow cytometry. An Annexin V-EGFP/PI Cell Apoptosis Detection Kit  was used for this experiment. Then, 0.25% trypsin without EDTA was used to digest cells, and the cells were centrifuged at 2000 rpm for 5 min. Pellets were washed twice with PBS. Binding buffer (500 μl) was added to suspend the cell pellets. After mixing the cells with 5 μl of Annexin V-FITC and 5 μl of propidium iodide and reacting for 5–15 min lucifugally, the cells were immediately analyzed using a FACSCalibur flow cytometer .p-value < 0.05 was considered statistically significant.All data were obtained from three independent experiments and are presented as the means ± SEM. All statistical analyses were performed using GraphPad Pro Prism 5.01 software . One-way ANOVA followed by the multiple comparisons test with the Bonferroni correction were employed to analyze the differences between sets of data. A In the diabetic mouse model induced by STZ, immunofluorescence staining was utilized to inspect P2X7R expression in the myocardial tissue of C57BL/6 mice . The expin vitro was consistent with the changes observed in vivo. As shown in We used HG (33 mM) to stimulate H9c2 cells at different time points and evaluate whether the upregulation of the P2X7R We performed additional experiments to detect the effect of P2X7R inhibition on the status and cardiac function of mice. The weight of C57BL/6 mice was decreased and fasting blood glucose levels increased significantly after the administration of STZ . HoweverNon-invasive transthoracic echocardiography was used to examine the cardiac function of all experimental mice 2 h before sacrifice . EchocarAlthough A438079 improved the cardiac function of diabetic mice, we were not sure whether it improved cardiac remodeling. Thus, H&E staining was used to detect the structural morphology. The hearts of STZ-challenged mice displayed structural abnormalities, such as disorganized myofibers. This disorder was improved in the other groups treated with the inhibitor .Fibrosis is also an important pathological variation observed in individuals with DCM . The conIn addition, apoptosis plays an important role in DCM. As shown in images of TUNEL staining, A438079 played a role in preserving myocardial cell survival and reducing apoptosis caused by diabetes . The levin vitro. H9c2 cells and primary rat cardiomyocytes were incubated with A438079 for 1 h prior to HG (33 mM) stimulation for 24 h. The protein levels of the hypertrophic marker MyHC and profibrotic proteins TGF-β, MMP-9, and COL-1 in the respective cells were remarkably decreased by the A438079 pretreatment (The tests described below were performed to confirm whether P2X7R inhibition using A438079 protected the myocardium reatment . The resreatment . As intureatment . These pin vitro to confirm the role of the P2X7R in HG-induced apoptosis. H9c2 cells and rat primary cardiomyocytes were pretreated with A438079 for 1 h and then exposed to HG (33 mM) for 24 h. As shown in A selective inhibitor of the P2X7R was used in vitro experiments, pharmacological inhibition of the myocardial P2X7R reduced cardiac remodeling and apoptosis in the STZ-induced model or HG-treated cells. P2X7R knockout mice (P2X7R–/–) were used as experimental models to better clarify its role. –/– mice.Based on previous animal experiments and –/– mice. All mice were subjected to echocardiography 2 h before sacrifice. A significant difference in the heart rate was not observed among the four groups. Under basal conditions, P2X7R knockout alone had no effect on cardiac function . As show–/– decreased cardiac fibrosis in STZ-induced mice  for 1 h and then incubated them with HG to further elucidate the relationship between PKCβ and ERK. After the addition of PKCβ and ERK inhibitors, the expression of the P2X7R did not change upon HG stimulation . In contin vivo and in vitro. We confirmed that the suppression of P2X7R inhibited PKCβ and ERK activation and then decreased the hallmarks of HG-induced myocardial remodeling. In addition, PKCβ was identified as an upstream molecule that regulates the activation of ERK in this process. Cardiovascular disease remains one of the main causes of death worldwide. In the present study, we systematically revealed the novel role of the P2X7R in DCM. The P2X7R was upregulated in the diabetic hearts and in HG-treated cardiomyocytes. The inhibition or knockout of the P2X7R significantly mitigated cardiac damage, including cardiomyocyte fibrosis, apoptosis, and hypertrophy, Diabetic cardiomyopathy is caused by prolonged diabetes . Diabetein vivo (–/– reversed these aberrant changes. Collectively, the P2X7R is involved in the process of fibrosis, hypertrophy, and apoptosis in heart tissues of diabetic mice, suggesting that the inhibition of P2X7R expression to prevent cardiac remodeling will be an effective cardioprotective strategy for abolishing DCM. Interestingly, the change in P2X7R expression only occurred in cardiomyocytes, but not in cultured rat primary fibroblasts (The P2X7R was first cloned from a rat brain . With adin vivo . In addiin vivo . Moreovein vivo . Thus, tin vivo . For exain vivo  and induin vivo . In the in vivo , 3. Cardin vivo , and redroblasts , which iroblasts . This ph2+ influx (Based on the P2X7R structure, the N terminus contains a consensus PKC phosphorylation site . Further+ influx , and ERK+ influx . In the + influx , indicat+ influx . Interes+ influx . Thus, P+ influx . Taken t+ influx .–/– mice. P2X7R inhibition or deficiency reduces myocardial hypertrophy, fibrosis, and apoptosis to subsequently improve cardiac function. Moreover, we further clarified that the P2X7R/PKCβ/ERK pathway is involved in the process and provided a new target for the treatment of DCM.In general, we described the role of the P2X7R in DCM using its specific inhibitors and P2X7RThe original contributions presented in the study are included in the article/The animal study was reviewed and approved by the Committee on Animal Care of Wenzhou Medical University.SH wrote the manuscript. SH and WW conceived, designed, and analyzed the data. SH, WW, and LL researched the data and performed the animal experiments. TW, YZ, and YL performed the cell experiments. WH reviewed and edited the manuscript. YW and ZH designed and supervised the study. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


SLC26A4 gene are globally a common cause of hearing impairment. In the past, cell lines and transgenic mice were widely used to investigate the pathogenicity associated with SLC26A4 variants. However, discrepancies in pathogenicity between humans and cell lines or transgenic mice were documented for some SLC26A4 variants. For instance, the p.C565Y variant, which was reported to be pathogenic in humans, did not exhibit functional pathogenic consequences in cell lines. To address the pathogenicity of p.C565Y, we used a genotype-based approach in which we generated knock-in mice that were heterozygous (Slc26a4+/C565Y), homozygous (C565Y/C565YSlc26a4), and compound heterozygous (919-2A>G/C565YSlc26a4) for this variant. Subsequent phenotypic characterization revealed that mice with these genotypes demonstrated normal auditory and vestibular functions, and normal inner-ear morphology and pendrin expression. These findings indicate that the p.C565Y variant is nonpathogenic for mice, and that a single p.C565Y allele is sufficient to maintain normal inner-ear physiology in mice. Our results highlight the differences in pathogenicity associated with certain SLC26A4 variants between transgenic mice and humans, which should be considered when interpreting the results of animal studies for SLC26A4-related deafness.Recessive variants of the  SLC26A4 (Gene ID: 5172) gene are a common global cause of hereditary hearing impairment (HHI) ; Tg[R]; Slc26a4Δ/Δ mice showed some residual hearing, neither could perfectly recapitulate the fluctuating or progressive disease nature in humans. Further studies are required to refine cell-line or transgenic-mouse models to investigate and better understand SLC26A4-related SNHI.Despite the presence of these correlations in the pathogenicity of ll lines ,9,37, orll lines . The trall lines , which c p.H723R ,11,12, in humans ,39. ThirSLC26A4 p.C565Y variant in humans in a genotype-driven approach. Subsequent phenotypic characterization revealed that mice with the Slc26a4 p.C565Y variant exhibit normal audiovestibular phenotypes and inner-ear morphology, indicating that the pathogenicity associated with specific SLC26A4 variants might differ between humans and mice. Therefore, with regard to research on SLC26A4-related SNHI, caution should be taken when translating results of animal studies to humans.In conclusion, we generated a knock-in mouse model that segregated the deafness-associated Slc26a4 gene and further generate the +/C565YSlc26a4 mouse strain. Specific guide RNAs (sgRNAs) were developed to target exon 15 of the Slc26a4 gene in the C57BL/6 mouse strain. The sgRNA and CRISPR/Cas9 RNA were delivered simultaneously into the zygote of a C57BL/6 mouse to generate the founders. Two male founder mice were obtained, each possessing the p.C565Y (c.1694G>A) variant in the Slc26a4 gene. After germline transmission of the targeted mutation allele, we were able to generate the congenic +/C565YSlc26a4 mouse line by repeated backcrossing with the C57BL/6 inbred strain for 6–10 generations. Thereafter, the homozygous mice for the Slc26a4p.C565Y variant (C565Y/C565YSlc26a4) were obtained by intercrossing heterozygous mice  and the clustered regularly interspaced short palindromic repeat (CRISPR) technology-associated RNA-guided endonuclease Cas9 to mutate the 919-2A>G/C565YSlc26a4) with both the p.C565Y and c.919-2A>G variants were also generated by intercrossing heterozygous +/C565YSlc26a4 mice with 919-2A>G/919-2A>GSlc26a4 mice [+/+Slc26a4, C565Y/C565YSlc26a4, 919-2A>G/C565YSlc26a4, and 919-2A>G/919-2A>GSlc26a4.Corresponding to the human genotypes, compound heterozygous mice  were intraperitoneally anesthetized with pentobarbital  and maintained in an acoustically and electrically insulated, and grounded test room . The thrFor vestibular evaluations, the mice were subjected to a series of tests , including a swimming test and the rotarod test. For the swimming test, the swimming performance of mice was scored from 0 to 3, with 0 being normal swimming and 3 being underwater tumbling . For theTo perform light-microscopy analysis, tissue samples were subjected to hematoxylin and eosin (H&E) staining, and the morphology of each sample was determined using a Leica optical microscope (Leica). First, inner-ear tissue from adult mice (3 months) was harvested and fixed using perilymphatic perfusion with 4% paraformaldehyde , prepared in phosphate-buffered saline (PBS). Specimens were decalcified for 1 week. Subsequently, samples were dehydrated and embedded in paraffin. Serial sections of 7 µm thickness were obtained and stained with H&E.For the whole-mount studies of mouse inner ears, specimens were prepared as previously described . Tissue 919-2A>G/C565YSlc26a4 and C565Y/C565YSlc26a4 mice. Tissue sections were mounted onto silane-coated glass slides, deparaffinized in xylene, and rehydrated in ethanol. Tissue samples were stained with rabbit antipendrin , and incubated with DAPI (1:5000) and Alexa Fluor 488-conjugated goat antirabbit IgG (H + L) secondary antibodies . After incubation, slides were washed with PBS and mounted with the ProLong Antifade Kit at 25 °C. Images were captured using a laser-scanning confocal microscope .For pendrin-expression analysis, tissue sections were harvested from the inner ears of p value < 0.05 indicates significance. All analyses were performed using SPSS/Windows software 15.0 .Data are presented as mean ± SD. Statistical analyses were conducted using unpaired Student’s t-test with Bonferroni correction for continuous variables. A