Journal: Advanced Science

Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging

doi: 10.1002/advs.77571

Figure Lengend Snippet: BMAd accumulation and sDPP4 release during skeletal aging activate BMM STING signaling. (a) Heatmap of dysregulated factors (≥1.5‐fold change) in the bone marrow supernatant of 3‐/18‐month‐old male rats identified through 4D label‐free technology ( n = 3 biological replicates). (b) Venn diagram of dysregulated factors in the bone marrow of rats. (c) ELISA of sDPP4 levels in conditioned medium collected from BMAds, Pre‐Ocs, BMMs, Ocs, BMSCs, Obs, and Neu ( n = 6 biological replicates; * p < 0.05 compared to BMAds‐CM). (d) Representative immunofluorescence staining showing that DPP4 was markedly detected in BMAds (Perilipin) in femora from 3‐/24‐month‐old male mice (scale bar: 100 µm). (e) ELISA of sDPP4 levels in conditioned medium collected in 3 M‐/18 M‐BMAds ( n = 6 biological replicates). (f,g) Representative micro‐CT images (f) and quantitative analysis of BV/TV, Tb. N, Tb.Th, and Tb.Sp (g) from PBS‐treated or rDPP4‐treated male mice ( n = 6 biological replicates). (h,i) Representative micro‐CT images (h) and quantitative analysis of BV/TV, Tb. N, Tb.Th, and Tb.Sp (i) from AAV9‐BMAd‐shNC‐treated or AAV9‐BMAd‐sh Dpp4 ‐treated male mice ( n = 6 biological replicates). (j) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs from PBS‐treated or rDPP4‐treated male mice ( n = 3 biological replicates). (k) Representative immunoblots of cGAS and STING protein/phosphorylation levels in BMMs treated with either rDPP4 alone or in combination with 18 M‐SN ( n = 3 biological replicates). Data are shown as mean ± SD.

Article Snippet: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Micro-CT, Analysis, Western Blot, Phospho-proteomics

Journal: Advanced Science

Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging

doi: 10.1002/advs.77571

Figure Lengend Snippet: BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and rTLR4. (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.

Article Snippet: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Derivative Assay, Binding Assay, Activation Assay, Western Blot, Phospho-proteomics, Docking Assay, Analysis, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Flow Cytometry, Cell Culture, Knockdown

Journal: Advanced Science

Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging

doi: 10.1002/advs.77571

Figure Lengend Snippet: STING signaling promotes NAD + decline in BMMs during skeletal aging by enhancing Cd38 transcription through IRF3 and P65. (a) WST‐8 analysis of NAD levels in 3, 10, 18, and 24 M‐BMMs ( n = 6 biological replicates). (b) Schematic of key enzymes in the NAD salvage biosynthesis pathway. (c,d) qPCR ( n = 6 biological replicates) and Western blotting ( n = 3 biological replicates) analyses of key enzymes in the NAD + salvage biosynthesis pathway in 3, 10, 18, and 24 M‐BMMs. (e) Fluorometric assay for CD38 NADase activity in 3 M‐/18 M‐BMMs ( n = 6 biological replicates). (f) WST‐8 analysis of NAD + levels in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (g,h) Fluorometric assay and WST‐8 analysis of CD38 NADase activity (g) or NAD + levels (h) in SN‐rDPP4‐treated BMMs with or without Cgas , Sting , Irf3, and Rela knockdown ( n = 6 biological replicates). (i) Representative immunoblots of CD38 protein levels in diABZI‐treated, SN‐rDPP4‐treated, or co‐cultured BMMs with or without Cgas , Sting , Irf3, and Rela knockdown ( n = 3 biological replicates). (j) Dual luciferase reporter assay in HEK 293T cells co‐transfected with CD38 luciferase reporter, IRF3 or P65 plasmid separately, and pRL‐CMV‐Renilla ( n = 6 biological replicates). (k) Chromatin immunoprecipitation assay showing that IRF3 or P65 bound to the CD38 promoter in BMMs. Binding levels were increased in BMMs treated with SN‐rDPP4 and were abolished by STING knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.

Article Snippet: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Analysis, Western Blot, Activity Assay, Knockdown, Cell Culture, Luciferase, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay

Journal: The Journal of infectious diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

doi: 10.1093/infdis/jiv499

Figure Lengend Snippet: Figure 1. Generation and characterization of K18-hDPP4 mice. A, The hDPP4 coding sequence was cloned into a plasmid containing the 5′ and 3′ genomic regions of human cytokeratin 18 (K18). The K18 5′ genomic region consists of a 2.5-kb upstream genomic sequence, promoter, and first intron of the human K18 gene while the K18 3′ region consists of exon 6, intron 6, exon 7, and approximately 300 base pairs of 3′ untranslated region of the human K18 gene, including the K18 polyA signal. Immediately upstream of the hDPP4 start codon is a translational enhancer (TE) sequence from alfalfa mosaic virus. B, Four K18-hDPP4 transgenic founder lines were generated and intranasally in- oculated with 1 × 105 plaque-forming units (PFU) of Middle East respiratory syndrome coronavirus. Lung titers of founder mice were determined by plaque assay 3 days after infection. Data are mean ± standard deviation [SD] for 6–10 mice/line. C, Quantitative measurement of human DPP4 concentrations in tissues of nontransgenic or K18-hDPP4 founder line 3 by enzyme-linked immunosorbent assay. Data are mean ± SD for 3 mice. Abbreviation: LOD, limit of detection.

Article Snippet: A FLAG epitope–tagged hDPP4 complementary DNA (Origene) was cloned into the pK18mTElacZ-K18i6×7pA vector to create pK18-hDPP4 [19–21].

Techniques: Sequencing, Clone Assay, Plasmid Preparation, Virus, Transgenic Assay, Generated, Plaque Assay, Infection, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of infectious diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

doi: 10.1093/infdis/jiv499

Figure Lengend Snippet: Figure 2. Middle East respiratory syndrome coronavirus (MERS-CoV) infection causes lethal disease in human cytokeratin 18–hDPP4 mice. A–C, Animals were inoculated intranasally with 1 × 105 plaque-forming units (PFU) of MERS-CoV and survival (A), weight (B), and temperature (C) monitored daily. Data are for 4 nontransgenic mice and 14 K18-hDPP4 mice. D, MERS-CoV titers in indicated tissues at days 2, 4, and 6 after infection. Data are for 3–4 mice. E, The copy numbers of viral RNA in indicated tissues at days 2 and 4 after infection were analyzed by quantitative real-time polymerase chain reaction targeting regions within open reading frame 1a. Data are for 4 mice. All results are expressed as mean ± SD.

Article Snippet: A FLAG epitope–tagged hDPP4 complementary DNA (Origene) was cloned into the pK18mTElacZ-K18i6×7pA vector to create pK18-hDPP4 [19–21].

Techniques: Infection, Real-time Polymerase Chain Reaction

Journal: The Journal of infectious diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

doi: 10.1093/infdis/jiv499

Figure Lengend Snippet: Figure 4. Brain disease in Middle East respiratory syndrome coronavirus (MERS-CoV)–infected human cytokeratin 18 (K18)–hDPP4 and uninfected mice. A, Normal brain from an uninfected mouse. B, MERS-CoV caused lymphocytic perivascular cuffing in the infected brain. C, Infected neuron in hippocampus 6 days after infection. Note the granular degeneration and basophilic cytoplasmic inclusions (arrow and inset). D, Dying cells undergoing degeneration (arrows and inset; 6 days after infection) are detected in highly infected regions such as the thalamus or brain stem. E, Meningeal and perivascular cuffing included neutrophilic infiltrates (arrows; 6 days after infection). F, Several degenerating cells had small to granular basophilic cytoplasmic inclusions (arrows; 6 days after infection) that were stained with anti–MERS-CoV antibody (inset; brown). Note the neuropil rarefaction. Section were stained with hematoxylin-eosin (original magnification ×600). G–I, Outcomes of K18-hDPP4 mice infected with different intranasal inocula of MERS-CoV. K18-hDPP4 mice received 1000, 100, or 10 plaque-forming units (PFU) of MERS-CoV and were monitored for survival (G) and weight (H). There were 5 mice/group. I, Lungs and brains of mice receiving 10 PFU were harvested 10 days after inoculation or when they lost 20% of body weight. A total of 3 of 7 MERS-CoV–infected mice showed high virus titers in the brains. J, MERS-CoV replicates in cells of the nervous system. Human central nervous system–derived cell lines (U-138 MG an SK-N-SH), primary porcine astrocytes, a murine astrocytoma cell line (DBT), and African green monkey kidney cells (Vero-81) were infected with MERS-CoV at a multiplicity of infection of 1. Titers from these cells immediately after infection (day 0) or 2 days after infection were determined by plaque assay. Data are mean ± SD for 3 replicates/condition. Abbreviation: LOD, limit of detection.

Article Snippet: A FLAG epitope–tagged hDPP4 complementary DNA (Origene) was cloned into the pK18mTElacZ-K18i6×7pA vector to create pK18-hDPP4 [19–21].

Techniques: Infection, Staining, Virus, Derivative Assay, Plaque Assay

Journal: The Journal of infectious diseases

Article Title: Middle East Respiratory Syndrome Coronavirus Causes Multiple Organ Damage and Lethal Disease in Mice Transgenic for Human Dipeptidyl Peptidase 4.

doi: 10.1093/infdis/jiv499

Figure Lengend Snippet: Figure 5. Immunization with Venezuelan equine encephalitis replicon particles (VRPs) expressing Middle East respiratory syndrome coronavirus (MERS-CoV) spike glyco- protein (VRP–MERS-S) or passive immunization protects human cytokeratin 18 (K18)–hDPP4 mice against MERS-CoV infection. A and B, K18-hDPP4 mice were immunized with 1 × 105 infectious units (IU) of VRPs expressing green fluorescent protein (VRP-GFP) or VRP-MERS-S in the footpad and boosted with the same dose 4 weeks later. Mice were infected with 1 × 105 plaque-forming units of MERS-CoV 2 weeks after the boost. C and D, For passive immunization, nontransgenic mice were immunized as described above. Sera were obtained 2 weeks after boosting and transferred into K18-hDPP4 mice intraperitoneally 1 day before infection with MERS-CoV. Survival and weights were recorded for active immunization (A and B) and passive immunization (C and D). Data are mean ± standard deviation (SD) for 5 mice/group. E and F, MERS-CoV titers in lung tissue (E) and brain tissue (F) 2, 4, and 6 days after infection in mice with or without passive immunization. Data are mean ± SD for 3 mice/group. Abbreviation: LOD, limit of detection.

Article Snippet: A FLAG epitope–tagged hDPP4 complementary DNA (Origene) was cloned into the pK18mTElacZ-K18i6×7pA vector to create pK18-hDPP4 [19–21].

Techniques: Expressing, Infection, Standard Deviation