recombinant mouse hevin  (R&D Systems)

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: The expression level of SPARCL1 in human OA cartilage. A Microarray profiling analysis data of SPARCL1 expression level in OA cartilage and normal cartilage. Date was originated from GSE113825. Unpaired T-test were used. B Immunohistochemistry assay with anti-SPARCL1 in undamaged and OA cartilage tissues. Scale bar, Left, 100 μm; Right, 50 μm. C Relative SPARCL1 expression level in OA (n = 55) and corresponding undamaged (n = 55) cartilage tissues based on an immunohistochemistry assay and significance was evaluated by paired Student's t test. Data are expressed as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques: Expressing, Microarray, Immunohistochemistry

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: Sparcl1 regulated ECM degradation in chondrocytes. A Alcian blue staining of ATDC5 with micromass culture in chondrogenesis media with or without recombinant Sparcl1 protein. Representative image of staining at day 8 is shown. B CCK-8 assay test of the cell viability of MCC treated with different concentration of recombinant Sparcl1 protein (10, 25, 50, 100 ng/ml). C Quantification of mRNA levels for Col2a1, Acan and Sox9 in MCC treated with recombinant Sparcl1 protein (10 and 50 ng/ml) for 48 h. D Quantification of mRNA levels for Mmp3, Mmp13, Adamts1, Adamts5 in MCC treated with recombinant Sparcl1 protein (10 and 50 ng/ml) for 48 h. E Quantification of mRNA levels for Col2a1, Acan, Mmp3, Mmp13, Ccl2 and IL-6 in MCC treated with IL-1β (10 ng/ml) and recombinant Sparcl1 protein (10, 25 and 50 ng/ml) for 48 h. Data are expressed as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with post-hoc Bonferroni correction was used for comparisons.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques: Staining, Recombinant, CCK-8 Assay, Concentration Assay

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: Sparcl1 accelerated OA process in ACLT mice model. A Representative safranin O-fast green images of osteoarthritic knee joints, which were collected 6 weeks after ACLT surgery. Yellow arrowheads indicated articular cartilage degradation. n = 8; Scale bar, left, 200 μm; right, 100 μm. B The severity of OA-like phenotype was analyzed using the Osteoarthritis Research Society International (OARSI) score system (n = 8). C The degree of synovitis was semi-quantified by the enlargement of synovial lining layers and density of cells (n = 8). D Three-dimensional models of mice knee joints. Red arrow showed osteophyte formation. Scale bar, 250 μm. E, F Osteophytes were semi-quantified by evaluating the osteophyte formation score consisting of two domains, size (E) and maturity (F) (n = 5) Data are expressed as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with post-hoc Bonferroni correction was used for comparisons.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques:

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: Sparcl1 exacerbated deteriorated subchondral bone in ACLT mice. A Representative micro-CT images of tibial plateau in coronal plane. B Representative 3D micro-CT images of subchondral trabecular bone of tibial plateau. C-J Quantitative micro-CT analysis of tibial subchondral trabecular bone: BV/TV (C), Tb. th (D), Tb.N (E), BS/TV (F), Tb. sp (G), Tb. Pf (H), BS/BV (I) and SMI (J). BV/TV, Bone Volume/Total Volume; Tb.Th, Trabecular Bone Number; Tb.N, Trabecular Bone Number; BS/TV, Bone Surface/Total Volume; Tb. Sp, Trabecular Bone Separation; Tb. Pf, Trabecular Bone pattern factor; BS/BV, Bone Surface/Bone Volume; SMI, structure model index. n = 5 in each group. Data are expressed as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with post-hoc Bonferroni correction was used for comparisons.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques: Micro-CT

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: RNA-seq analysis of Sparcl1 treated chondrocytes. A A volcano plot illustrating differentially regulated gene expression from RNA-seq analysis between ctrl and Sparcl1 treated MCCs. Genes upregulated and downregulated are shown in red and blue, respectively. B The Gene Ontology (GO) functional clustering of genes in Sparcl1-treated MCC (top 20 most significantly affected categories are shown) C-D Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of total genes (C) and upregulated genes (D) in Sparcl1-treated MCC transcriptome. E Pathway network illustrated the relationships of each pathway enriched in KEGG analysis.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques: RNA Sequencing, Gene Expression, Functional Assay

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: Inflammatory response was increased in Sparcl1 treated chondrocytes. A GSEA plots evaluating the changes of inflammatory response related genes. B STRING analysis identified protein–protein interactions in the enriched pathways after Sparcl1 stimulation. C Quantification of mRNA levels for DEGs mentioned above in chondrocytes treated with Sparcl1 protein (10, 25, 50 ng/ml). Data are expressed as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with post-hoc Bonferroni correction was used for comparisons.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques: Protein-Protein interactions

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: Sparcl1 regulated inflammation and ECM degradation through TNF/NF-κB pathway. A GSEA plots evaluating the changes of ECM disassembly related genes. B Protein expression of FAK, pFAK, ADAMTS5 and MMP3 in chondrocytes treated with recombinant Sparcl1 protein (10, 25, 50 ng/ml) for 72 h analyzed by Western blot, and quantification of immunoblots (n = 3). C GSEA plots evaluating the changes of TNFA signaling via NF-κB related genes. D-E Protein expression of TNF, IKKα, IKKβ, and NF-κB in chondrocytes treated with recombinant Sparcl1 protein (10, 25, 50 ng/ml) for 72 h analyzed by Western blot, and quantification of immunoblots (n = 3). Data are expressed as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001. One-way ANOVA with post-hoc Bonferroni correction was used for comparisons.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques: Expressing, Recombinant, Western Blot

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: BAY 11 – 7082 could reverse Sparcl1 induced ECM degradation and related gene expression. A Alcian blue staining of ATDC5 with micromass culture in chondrogenesis media with or without recombinant Sparcl1 protein and BAY 11–7082 (2.5 μM). Representative image of staining at day 8 is shown. B Quantification of mRNA levels for Col2a1, Acan, Sox9, Mmp3, Mmp13, Adamts5 in MCC treated with recombinant Sparcl1 protein (50 ng/ml) and BAY 11–7082 (2.5 μM) for 48 h. C Immunoblot analysis of MCC, immunoprecipitated with anti-TNFR and IgG and analyzed by immunoblot with anti-SPARCL1.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques: Gene Expression, Staining, Recombinant, Western Blot, Immunoprecipitation

Journal: Journal of Orthopaedic Translation

Article Title: SPARCL1 promotes chondrocytes extracellular matrix degradation and inflammation in osteoarthritis via TNF/NF-κB pathway

doi: 10.1016/j.jot.2024.02.009

Figure Lengend Snippet: Mechanism of Sparcl1 in exacerbate osteoarthritis. Diagram illustrating the mechanism how Sparcl1 influences OA pathogenesis through the TNF/NFκB signaling pathway.

Article Snippet: Cells were seeded onto 96-well plates (1,0000 cells per well) and treated with recombinant mouse Sparcl1 protein (R&D Systems Inc.).

Techniques:

Journal: Nature Communications

Article Title: Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation

doi: 10.1038/s41467-024-48589-3

Figure Lengend Snippet: A Schematic of mouse lung endothelial single-cell sequencing preparation. B ScRNA-seq analysis for mouse lung ECs sorted from uninjured (D0) and on 20 and 30 days after influenza infection (marked as D20 and D30, respectively). Uniform manifold approximation and projection (UMAP) plots showing the dynamics in gCap ECs. C The 2 gCap EC clusters of interest, cluster_0 (Dev.ECs) and cluster_1 (Immu.ECs), were subsetted from ( B ). D Heatmap showing the top 20 differentially expressed genes of cluster_0 (Dev.ECs) and cluster_1(Immu.ECs). E UMAP analysis reveals that Sparcl1 is predominantly expressed in gCap ECs, especially in Immu.ECs. F Violin plots showing Sparcl1 expression level in mouse lung ECs sorted from D0, D20 and D30, respectively. G Representative immunostaining of SPARCL1 in endothelial cells (CD31) in both uninjured (D0) and D20 after influenza infection lung tissues. Scale bar, 100 μm. H qPCR analysis of Sparcl1 in isolated lung ECs (CD45 − CD31 + ) sorted on days 0 (uninjured), 20 and 27 after influenza infection, n = 3–4 mice per group (each dot represents one mouse), independent biological replicates. D0 vs. D20: p = 0.006; D0 vs. D27: p = 0.044. I The concentration of SPARCL1 in bronchoalveolar lavage fluid (BALF) was measured by ELISA at 0 (uninjured), 15, 20, and 30 days after influenza infection, n = 4 mice per group, independent biological replicates. D0 vs. D15: p < 0.0001; D0 vs. D20: p = 0.0001. Data in H and I are presented as means ± SEM, calculated using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test. * P < 0.05, ** P < 0.01, *** P < 0.001 and **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Depending on the experiment, BMDMs were treated with recombinant mouse SPARCL1 (0–20 μg/ml, R&D system, #4547-SL; SinoBiological, #50544-M08H); recombinant IL-4 (20 ng/ml, PeproTech, #214-14), lipopolysaccharides (50 ng/ml; Sigma Aldrich, #L6529), Resatorvid (TAK-242) (10 μM; MedChemExpress, #HY-11109), LPS-EB Ultrapure (LPS from E. coli O111:B4, InvivoGen, #tlrl-3pelps) or vehicle control.

Techniques: Sequencing, Infection, Expressing, Immunostaining, Isolation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation

doi: 10.1038/s41467-024-48589-3

Figure Lengend Snippet: A Time course of changes in body weight and B capillary oxygen saturation in WT and EC Sparcl1-KO mice after influenza infection, n = 7 mice per group, independent biological replicates. A p = 0.04; B p = 0.04. C Kaplan–Meier survival curves after influenza infection, log-rank test. D–H The levels of pro-inflammatory cytokines IL-6 ( D ), TNF ( E ), IFN-γ( F ), IL-4( G ), and IL-10 ( H ) in bronchoalveolar lavage fluid (BALF) were measured by ELISA in WT and EC Sparcl1-KO mice at day 0 (uninjured) and/or D12 days after influenza infection, each dot represents one mouse, n = 4–7 mice per group, independent biological replicates. D p = 0.03; E p = 0.04. I and J Total protein ( I ) and cells ( J ) were quantified in BALF on day 12 post influenza infection, n = 6 mice per group, I p = 0.018, J p = 0.07. K CCL-2 concentration in BALF from WT (wild-type) and EC Sparcl1-KO mice at day 12 after infection. n = 6 mice per group, independent biological replicates, p = 0.02. L . Quantification of the proportion of total macrophages (CD64 + F4/80 + ), alveolar macrophages (CD45 + Ly6G - CD64 + F4/80 + SiglecF + ) and interstitial and recruited macrophages (CD45 + Ly6G - CD64 + F4/80 + SiglecF - ) in CD45 + live cells at day 12 after influenza infection in WT (n = 7 mice) and EC Sparcl1-KO (n = 6 mice) mice, independent biological replicates. Gated as shown in Supplementary Fig. . Total macrophages: WT vs. EC Sparcl1-KO : p = 0.033. M Left: tile scan images of H&E stain at 25 days post-infection, demarcated boxes indicate different injury zones. Right: clustered injury zone maps produced from left H&E images, scale bars, 1 mm. N Quantification of injured area in different injury zones in M , n = 5 mice per group, independent biological replicates. Total injured zone: WT vs. EC Sparcl1-KO : p = 0.033. Data in A , B , D to ( L ) and ( N ) are presented as means ± SEM, calculated using unpaired two-tailed t -test. Data in ( C ) were calculated using log-rank test. * P < 0.05, ns not significant, P > 0.05. Source data are provided as a Source Data file.

Article Snippet: Depending on the experiment, BMDMs were treated with recombinant mouse SPARCL1 (0–20 μg/ml, R&D system, #4547-SL; SinoBiological, #50544-M08H); recombinant IL-4 (20 ng/ml, PeproTech, #214-14), lipopolysaccharides (50 ng/ml; Sigma Aldrich, #L6529), Resatorvid (TAK-242) (10 μM; MedChemExpress, #HY-11109), LPS-EB Ultrapure (LPS from E. coli O111:B4, InvivoGen, #tlrl-3pelps) or vehicle control.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Staining, Produced, Two Tailed Test

Journal: Nature Communications

Article Title: Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation

doi: 10.1038/s41467-024-48589-3

Figure Lengend Snippet: A Schematic of the strategy used to generate Sparcl1 knock-in mice targeting ROSA26 locus. B DNA gel image for genotyping of endothelial Sparcl1 knock-in mice. Lanes a and b represent wild-type mice with PCR product at 145 bp. Lanes c and d represent heterozygous mice with product at 145 bp (wt) and product at 104 bp (mut). Lanes e and f represent homozygous mice with PCR product at 104 bp. C Western blot of SPARCL1 in whole lung tissue from EC Sparcl1-OE (VECad CreERT2 ; Sparcl1 +/wt or VECad CreERT2 ; Sparcl1 +/+ ) or WT ( VECad CreERT2 ) mice 2 weeks after 5 doses of tamoxifen, the image showing representative data from n = 4 biological replicates. D Time course of changes in body weight and E capillary oxygen saturation in WT and EC Sparcl1-OE mice after influenza infection. D WT: n = 9 mice, EC Sparcl1-OE : n = 7 mice, independent biological replicates; D19: p = 0.02, D23: p = 0.03. E n = 5 mice per group, independent biological replicates; D20: p = 0.03. F Kaplan–Meier survival curves after influenza infection, log-rank test. WT: n = 12 mice, EC Sparcl1-OE n = 14 mice. Total protein ( G ) and cells ( H ) were quantified in BALF on day 20 post influenza infection, n = 6 mice per group, independent biological replicates. G p = 0.02; H p = 0.046. I The concentration of pro-inflammatory cytokines, IL-1β, IL-6 and TNF in BALF were measured by ELISA in WT and EC Sparcl1-OE mice at 0 (uninjured, n = 3 independent biological replicates per group), 10 ( n = 5 independent biological replicates per group) and 20 ( n = 5 independent biological replicates per group) days post-influenza infection. IL-1β (D20): p = 0.037; IL-6 (D20): p = 0.04; TNF (D20): p = 0.032. ELISA measurement of IFN-γ ( J ), IL-4 ( K ), IL-10 ( L ), and CCL-2 ( M ) in WT and EC Sparcl1-OE mice on day 20 post-infection. n = 5 mice per group, independent biological replicates. CCL-2: p = 0.017. N . Quantification of the proportion of total macrophages (CD64 + F4/80 + ), alveolar macrophages (CD45 + Ly6G - CD64 + F4/80 + SiglecF + ) and interstitial and recruited macrophages (CD45 + Ly6G - CD64 + F4/80 + SiglecF - ) in CD45 + live cells at day 20 after influenza infection in WT and EC Sparcl1-OE mice, n = 5 mice per group, independent biological replicates. Gated as shown in Supplementary Fig. . Total macrophages: p = 0.037; interstitial and recruited macrophages: p = 0.032. Data in D , E and G to H and J to N are presented as means ± SEM, calculated using an unpaired two-tailed t -test. Data in I is presented as means ± SD, calculated using an unpaired two-tailed t -test. * P < 0.05. Data in F were calculated using log-rank test. * P < 0.05, ns: not significant, P > 0.05. Source data are provided as a Source Data file.

Article Snippet: Depending on the experiment, BMDMs were treated with recombinant mouse SPARCL1 (0–20 μg/ml, R&D system, #4547-SL; SinoBiological, #50544-M08H); recombinant IL-4 (20 ng/ml, PeproTech, #214-14), lipopolysaccharides (50 ng/ml; Sigma Aldrich, #L6529), Resatorvid (TAK-242) (10 μM; MedChemExpress, #HY-11109), LPS-EB Ultrapure (LPS from E. coli O111:B4, InvivoGen, #tlrl-3pelps) or vehicle control.

Techniques: Knock-In, Western Blot, Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation

doi: 10.1038/s41467-024-48589-3

Figure Lengend Snippet: A Representative gating scheme for identification of pulmonary M1-like (CD86 + CD206 − ) and M2-like (CD86 − CD206 + ) macrophages (CD45 + Ly6G − CD64 + F4/80 + ) at day 20 after influenza infection in WT and EC Sparcl1-OE mice. Macrophage gating strategies as shown in Supplementary Fig. . Quantification of the proportion of B M1-like (CD86 + CD206 − ) and C M2-like (CD86 − CD206 + ) macrophages in total lung macrophages (CD45 + Ly6G − CD64 + F4/80 + ), alveolar macrophages (CD45 + Ly6G − CD64 + F4/80 + SiglecF + ) and interstitial and recruited macrophages (CD45 + Ly6G − CD64 + F4/80 + SiglecF − ) at day 20 after influenza infection in WT and EC Sparcl1-OE mice, n = 5 mice per group, independent biological replicates. WT vs. EC Sparcl1-OE mice: B p = 0.005(Total MΦ), p = 0.015(Alveolar MΦ), p = 0.01(Interstitial+ recruited MΦ); C p = 0.002(Total MΦ), p = 0.026(Alveolar MΦ), p = 0.019(Interstitial+ recruited MΦ). D Gating strategy for M2-like macrophage (RELMα + ) in WT and EC Sparcl1-OE mice on day 20 post influenza infection. E Quantification of M2-like macrophage (RELMα + /CD64 + F4/80 + ) by flow cytometry analysis in WT ( n = 5 independent biological replicates) and EC Sparcl1-OE ( n = 6 independent biological replicates) mice, p = 0.0031. F Left: representative immunostaining of lung M2_like macrophage (RELMα + F4/80 + ) in WT and EC Sparcl1-OE mice on day 20 post influenza infection, scale bars, 25 μm. Right: quantification of the proportion of M2_like macrophages (RELMα + F4/80 + /F4/80 + ) in left, n = 3 mice per group, independent biological replicates, p = 0.038. G Principal components analysis (PCA) indicates transcriptomic changes in lung macrophages between WT ( n = 2 independent biological replicates) and EC Sparcl1-OE mice ( n = 3 independent biological replicates) on day 20 post-influenza infection. H Gene set enrichment analysis (GSEA) of RNA-seq profiles of purified lung macrophages isolated from WT and EC Sparcl1-OE mice on day 20 post influenza infection. Enrichment score and p value are displayed. I Volcano plot indicating the M1-associated genes upregulated in lung macrophage from EC Sparcl1-OE mice and M2-associated genes down-regulated on day 20 post influenza infection. Data in B , C , and E are presented as means ± SEM, and data in F is presented as means ± SD, calculated using an unpaired two-tailed t -test. * P < 0.05, ** P < 0.01. Source data are provided as a Source Data file.

Article Snippet: Depending on the experiment, BMDMs were treated with recombinant mouse SPARCL1 (0–20 μg/ml, R&D system, #4547-SL; SinoBiological, #50544-M08H); recombinant IL-4 (20 ng/ml, PeproTech, #214-14), lipopolysaccharides (50 ng/ml; Sigma Aldrich, #L6529), Resatorvid (TAK-242) (10 μM; MedChemExpress, #HY-11109), LPS-EB Ultrapure (LPS from E. coli O111:B4, InvivoGen, #tlrl-3pelps) or vehicle control.

Techniques: Infection, Flow Cytometry, Immunostaining, RNA Sequencing Assay, Purification, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation

doi: 10.1038/s41467-024-48589-3

Figure Lengend Snippet: A Western blotting analysis of indicated proteins in bone marrow-derived macrophages (BMDMs) treated with different doses of recombinant SPARCL1 protein (0–20 μg/ml) for 1 h, representative image from n = 3 biological replicates. B BMDMs were treated with different doses of recombinant SPARCL1 protein (0–20 μg/ml, Sino Biological) for 24 h, and cell supernatant was collected. Pro-inflammatory cytokines IL-1β, IL-6 and TNF levels in supernatant were measured by ELISA, n = 3 biological replicates per group. IL-1β: p = 0.01 (Control vs. 5), p = 0.0003 (Control vs. 10), p < 0.0001 (Control vs. 20); IL-6: p = 0.0005 (Control vs. 5), p < 0.0001 (Control vs.10), p < 0.0001 (Control vs. 20); TNF: p < 0.0001 (Control vs. 5), p < 0.0001 (Control vs. 10), p < 0.0001 (Control vs. 20). C Schematic of SPARCL1 treatment of BMDMs. D Quantification of the proportion of M2-like (F4/80 + CD206+) macrophages in M2 polarized BMDMs after being treated with SPARCL1 (10 μg/ml, Sino Biological) for 24 h. n = 3 biological replicates per group. M0 + PBS vs. M2 + PBS: p < 0.001; M2 + PBS vs. M2 + SPARCL1: p = 0.003. E qPCR analysis for M2 macrophage genes ( Chil3 and Mrc1 ) in M1 and M2 polarized BMDMs after being treated with SPARCL1 (10 μg/ml) for 24 h, n = 3 biological replicates per group. Mrc1: M2 + PBS vs. M2 + SPARCL1: p = 0.021; Chil3: M2 + PBS vs. M2 + SPARCL1: p = 0.0098. F BMDMs were pre-treated with TLR4 inhibitor, TAK-242(10 μM) for 1 h and then incubated with or without SPARCL1 (10 μg/ml, Sino Biological) for 1 h. Phosphorylation of NF-κB was detected by western blot assay, n = 3 biological replicates per group. G BMDMs were pre-treated with TLR4 inhibitor, TAK-242(10 μM) for 1 h and then incubated with or without SPARCL1 (10 μg/ml, Sino Biological) for 24 h, and cell supernatant was collected. Pro-inflammatory cytokines IL-1β, IL-6 and TNF levels in supernatant were measured by ELISA, n = 3 biological replicates per group. IL-1β (Control vs. SPARCL1: p = 0.0002; SPARCL1 vs. SPARCL1 + TAK-242: p = 0.006); IL-6 (Control vs. SPARCL1: p < 0.0001; SPARCL1 vs. SPARCL1 + TAK-242: p < 0.0001); TNF (Control vs. SPARCL1: p < 0.0001; SPARCL1 vs. SPARCL1 + TAK-242: p < 0.0001). H GSEA analysis indicates that endothelial overexpression of Sparcl1 in vivo positively engaged the TLR4 signaling in isolated macrophages (see Fig. ). I The TLR4 inhibitor TAK-242 blocks SPARCL1-induced NF-κB activation, thereby inhibiting macrophage transition to a pro-inflammatory phenotype. Data in B , D , and G are presented as means ± SEM, calculated using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test. Data in E are presented as means ± SEM, calculated using unpaired two-tailed t -test; * P < 0.05, ** P < 0.01, *** P < 0.001. Source data are provided as a Source Data file. Schematics and icons created with BioRender.com.

Article Snippet: Depending on the experiment, BMDMs were treated with recombinant mouse SPARCL1 (0–20 μg/ml, R&D system, #4547-SL; SinoBiological, #50544-M08H); recombinant IL-4 (20 ng/ml, PeproTech, #214-14), lipopolysaccharides (50 ng/ml; Sigma Aldrich, #L6529), Resatorvid (TAK-242) (10 μM; MedChemExpress, #HY-11109), LPS-EB Ultrapure (LPS from E. coli O111:B4, InvivoGen, #tlrl-3pelps) or vehicle control.

Techniques: Western Blot, Derivative Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Over Expression, In Vivo, Isolation, Activation Assay, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation

doi: 10.1038/s41467-024-48589-3

Figure Lengend Snippet: A Timeline for TAK-242 administration and sampling. WT and EC Sparcl1-OE mice were treated with TAK-242 (3 mg/kg, i.p.) or equal volume of vehicle control (DMSO). B and C Time course of changes in ( B ) body weight and ( C ) capillary oxygen saturation in EC Sparcl1-OE mice treated with or without TAK-242 after influenza infection. B n = 6 mice per group, independent biological replicates; D15: Control vs. TAK-242: p = 0.02; C Control: n = 5 mice, independent biological replicates, TAK-242: n = 6 mice, independent biological replicates; D15: Control vs. TAK-242: p = 0.018. D Representative gating scheme for identification of M1-like (CD86 + CD206 − ) and M2-like (CD86 − CD206 + ) macrophages in total lung macrophages (CD45 + Ly6G − CD64 + F4/80 + ), alveolar macrophages (CD45 + Ly6G − CD64 + F4/80 + SiglecF + ), and interstitial and recruited macrophages (CD45 + Ly6G − CD64 + F4/80 + SiglecF − ) at day 20 after influenza infection in EC Sparcl1-OE mice treated with or without TAK-242, Macrophage gating strategies as shown in Supplementary Fig. . E and F Quantification of the proportion of E M1-like (CD86 + CD206 − ) and F M2-like (CD86 − CD206 + ) macrophages in total lung macrophages (CD45 + Ly6G − CD64 + F4/80 + ), alveolar macrophages (CD45 + Ly6G − CD64 + F4/80 + SiglecF + ) and interstitial and recruited macrophages (CD45 + Ly6G − CD64 + F4/80 + SiglecF − ) at day 20 after influenza infection in WT and EC Sparcl1-OE mice treated with or without TAK-242, n = 5 mice per group, independent biological replicates. E p = 0.04 (total MΦ), p = 0.038 (alveolar MΦ). F p = 0.045 (interstitial + recruited MΦ). G The levels of pro-inflammatory cytokines, IL-1β, IL-6 and TNF in bronchoalveolar lavage fluid (BALF) were measured by ELISA in WT and EC Sparcl1-OE mice treated with or without TAK-242 at day 20 after influenza infection, n = 5 mice per group, independent biological replicates. IL-1β (Control: WT vs. EC Sparcl1-OE : p = 0.048; EC Sparcl1-OE mice: Control vs. TAK-242: p = 0.013); IL-6 ( EC Sparcl1-OE mice: Control vs. TAK-242: p = 0.023); TNF (WT mice: Control vs. TAK-242: p = 0.011; EC Sparcl1-OE mice: Control vs. TAK-242: p = 0.033). Data in B , C , E , and F are presented as means ± SEM, calculated using unpaired two-tailed t -test; Data in G are presented as means ± SEM, calculated using one-way analysis of variance (ANOVA), followed by Dunnett’s multiple comparison test. * P < 0.05. Source data are provided as a Source Data file.

Article Snippet: Depending on the experiment, BMDMs were treated with recombinant mouse SPARCL1 (0–20 μg/ml, R&D system, #4547-SL; SinoBiological, #50544-M08H); recombinant IL-4 (20 ng/ml, PeproTech, #214-14), lipopolysaccharides (50 ng/ml; Sigma Aldrich, #L6529), Resatorvid (TAK-242) (10 μM; MedChemExpress, #HY-11109), LPS-EB Ultrapure (LPS from E. coli O111:B4, InvivoGen, #tlrl-3pelps) or vehicle control.

Techniques: Sampling, Infection, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation

doi: 10.1038/s41467-024-48589-3

Figure Lengend Snippet: A Representative immunostaining image of SPARCL1 in endothelial cells (ERG) in both healthy and COVID-19 donors’ lung tissue. Scale bar, 50 μm. B qPCR analysis of SPARCL1 in isolated lung ECs (CD45 − EpCAM − CD31 + ) sorted from both healthy ( n = 5) and COVID-19 (n = 4) donors’ lung tissue, p = 0.013. C The SPARCL1 level in plasma from COVID-19 patients was measured by Olink proximal extension assay. For each group, the box depicts the median (center line) and upper and lower quartile. Upper quartile = 75th percentile and lower quartile = 25th percentile of the data. Data are presented as means ± SEM, Data in B were calculated using unpaired two-tailed t -test; Data in C were calculated using 2-sided, Wilcoxon Rank sum test comparing 90-day survivors ( n = 131) to 90-day non-survivors ( n = 28), * P < 0.05. Source data are provided as a Source Data file.

Article Snippet: Depending on the experiment, BMDMs were treated with recombinant mouse SPARCL1 (0–20 μg/ml, R&D system, #4547-SL; SinoBiological, #50544-M08H); recombinant IL-4 (20 ng/ml, PeproTech, #214-14), lipopolysaccharides (50 ng/ml; Sigma Aldrich, #L6529), Resatorvid (TAK-242) (10 μM; MedChemExpress, #HY-11109), LPS-EB Ultrapure (LPS from E. coli O111:B4, InvivoGen, #tlrl-3pelps) or vehicle control.

Techniques: Immunostaining, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Vascular endothelial-derived SPARCL1 exacerbates viral pneumonia through pro-inflammatory macrophage activation

doi: 10.1038/s41467-024-48589-3

Figure Lengend Snippet: In influenza pneumonia, SPARCL1 from endothelial cells enters the alveoli via damaged blood vessels, prompting alveolar macrophages to adopt a pro-inflammatory (M1-like) state. This transformation hinges on SPARCL1 inducing TLR-4/NF-κB signaling activation in alveolar macrophages, leading to increased expression of pro-inflammatory cytokines. Simultaneously, heightened CCL-2 attracts more monocytes and macrophages into the alveoli. These recruited macrophages, once again exposed to SPARCL1 within the alveoli, further adopt a pro-inflammatory phenotype. The cumulative effect intensifies the local inflammatory response, causing tissue damage and hindering lung repair. Schematics and icons created with BioRender.com.

Article Snippet: Depending on the experiment, BMDMs were treated with recombinant mouse SPARCL1 (0–20 μg/ml, R&D system, #4547-SL; SinoBiological, #50544-M08H); recombinant IL-4 (20 ng/ml, PeproTech, #214-14), lipopolysaccharides (50 ng/ml; Sigma Aldrich, #L6529), Resatorvid (TAK-242) (10 μM; MedChemExpress, #HY-11109), LPS-EB Ultrapure (LPS from E. coli O111:B4, InvivoGen, #tlrl-3pelps) or vehicle control.

Techniques: Transformation Assay, Activation Assay, Expressing