recombinant mouse il 10 protein  (R&D Systems)

Journal: Biomaterials Research

Article Title: Tryptophan-Enriched Lactobacillus rhamnosus GG-derived Nanovesicles Promote Alveolar Bone Regeneration through Macrophage Fatty Acid Oxidation

doi: 10.34133/bmr.0370

Figure Lengend Snippet: Isolation, characterization, cellular uptake of Lactobacillus rhamnosus GG-derived extracellular vesicles (LEVs) and phenotypic analysis of LEV-treated macrophages. (A) Transmission electron microscopy (TEM) image of isolated LEVs, showing their shapes with a double-layered membrane structure. (B) Western blot analysis showing the bacterial origin of LEVs. An anti-lipoteichoic acid (anti-LTA) antibody was used to detect the presence of LTA in LEVs and their parental bacteria L. rhamnosus GG ( LGG ) lysate, with the phosphate-buffered saline (PBS) group serving as a negative control. (C) Nanoparticle tracking analysis (NTA) graph showing the particle size distribution of isolated LEVs. (D) Immunofluorescence images showing the uptake of 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO)-labeled LEVs (green) by gingival macrophages (red, F4/80 + ) 24 h after injection into the periodontal tissue of a mouse model. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (E) In vitro immunofluorescence images showing the uptake of DiO-labeled LEVs (green) by macrophages (membrane stained red with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate [DiI]). Nuclei were stained with DAPI (blue). (F) Volcano plot showing differentially expressed genes (DEGs) in LEV-treated macrophages compared to control cells. (G) Bar plot showing the expression levels of M1 and M2 macrophage signature genes in LEV-treated cells. (H) Gene Ontology enrichment analysis of the DEGs, revealing enrichment in terms related to vesicle recognition, cell polarization and anti-inflammatory regulation, metabolic reprogramming, phagocytosis and apoptotic cell clearance, signal transduction, and protein regulation. (I) Cell Counting Kit-8 (CCK-8) assay results showing the effect of different concentrations of LEVs on the viability of macrophages challenged with lipopolysaccharide (LPS). (J) Western blot analysis showing the protein expression levels of M2 polarization markers (arginase-1 [ARG1], CD206, CD163, and interleukin-10 [IL-10]) in macrophages treated with different concentrations of LEVs. (K) Western blot analysis of the expression levels of key fatty acid oxidation (FAO) enzymes in LEV-treated macrophages. (L) Western blot analysis demonstrating the time-dependent effect of LEVs on carnitine palmitoyltransferase 1A (CPT1A) protein expression in macrophages. Additionally, a control group treated with heat-inactivated LGG (56 °C for 30 min) is included. * P < 0.05, ** P < 0.01, and **** P < 0.0001 in the indicated groups.

Article Snippet: The recombinant mouse IL-10 (rmIL-10) protein was purchased from ABclonal Technology (no. RP01465).

Techniques: Isolation, Derivative Assay, Transmission Assay, Electron Microscopy, Membrane, Western Blot, Bacteria, Saline, Negative Control, Immunofluorescence, Labeling, Injection, Staining, In Vitro, Control, Expressing, Transduction, Cell Counting, CCK-8 Assay

Journal: Biomaterials Research

Article Title: Tryptophan-Enriched Lactobacillus rhamnosus GG-derived Nanovesicles Promote Alveolar Bone Regeneration through Macrophage Fatty Acid Oxidation

doi: 10.34133/bmr.0370

Figure Lengend Snippet: Metabolic profiling and comparative analysis of glycolysis and fatty acid oxidation (FAO) in periodontitis tissues. (A) Single-sample gene set enrichment analysis (ssGSEA) method scores of metabolic pathways and immune-related signatures in periodontitis and healthy tissue samples. The heatmap shows the relative enrichment of glycolysis, inflammatory response, M1/M2 macrophage ratio, and FAO pathways. (B) Boxplots showing the statistical difference in the enrichment scores for each pathway between the healthy control and periodontitis groups. (C) Scatter plots with bivariate linear regression analysis showing the correlation between inflammatory response and FAO (left) as well as between inflammatory response and glycolysis (right). The correlation coefficient ( r ) and P value for each correlation are indicated. (D) Representative reconstructed and 3-dimensional (3D) scanned sections along the longitudinal direction of the maxilla. The distance (mm) between the cementoenamel junction (CEJ) and the alveolar bone crest (ABC) and the percentage of bone volume/total volume (BV/TV) were analyzed. BV/TV represents the bone volume fraction, that is, bone volume over tissue volume. (E) Western blot analysis of macrophage polarization markers (CD163, CD86, arginase-1 [ARG1], interleukin-10 [IL-10], and interleukin-6 [IL-6]) in periodontitis tissue, with or without etomoxir treatment, and in the phosphate-buffered saline (PBS) control group. (F) The expression of CD163, IL-10, carnitine palmitoyltransferase 1A (CPT1A), ARG1, IL-6, inducible nitric oxide synthase (iNOS), and CD86 in RAW264.7 cells stimulated with Porphyromonas gingivalis (multiplicity of infection [MOI] 100) were detected by Western blot analysis. (G) The expressions of iNOS, IL-6, CD86, IL-10, and CPT1A in RAW264.7 cells stimulated with increasing concentrations of Pg LPS (50, 100, 200, and 500 ng/ml) were detected by Western blot analysis. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 in the indicated groups.

Article Snippet: The recombinant mouse IL-10 (rmIL-10) protein was purchased from ABclonal Technology (no. RP01465).

Techniques: Control, Western Blot, Saline, Expressing, Infection

Journal: Biomaterials Research

Article Title: Tryptophan-Enriched Lactobacillus rhamnosus GG-derived Nanovesicles Promote Alveolar Bone Regeneration through Macrophage Fatty Acid Oxidation

doi: 10.34133/bmr.0370

Figure Lengend Snippet: Evaluation of macrophage polarization, alveolar bone, and carnitine palmitoyltransferase 1A (CPT1A)-mediated fatty acid oxidation (FAO) following Lactobacillus rhamnosus GG-derived extracellular vesicle (LEV) treatment. (A) Representative micro-computed tomography (micro-CT) images of the maxillary alveolar bone, evaluating bone loss in the periodontitis and LEV-treated groups. (B) Histological analysis of periodontal tissue sections with hematoxylin and eosin (H&E) staining. (C) Immunofluorescence images of periodontal tissue, showing macrophage infiltration and the labeling of CD86 + M1 macrophages. (D) Immunofluorescence images showing the labeling of CD163 + M2 macrophages and the expression of CPT1A in periodontal tissue. (E) Representative flow cytometric plots were used to evaluate the proportion of CD163 + M2 macrophage (F4/80 + ) subset in periodontal tissues. (F) The expressions of CPT1A, CD206, CD86, CD163, arginase-1 (ARG1), and interleukin-10 (IL-10) in the gingiva of mice were detected by Western blot analysis. (G to I) RAW264.7 cells were exposed to lipopolysaccharide (LPS), with or without LEV treatment. (G) Representative immunofluorescence images showing the morphology of macrophages and the expression of CD163 and CPT1A after different treatments. (H) Western blot analysis of macrophage polarization markers (CD163, ARG1, IL-10, and CPT1A) in RAW264.7 cells after LPS and LEV cotreatment. (I) Flow cytometry analysis to assess the proportions of different macrophage subsets (CD86 + , CD163 + , and CD206 + ) after LEV treatment. (J) Western blot analysis showing the protein expression levels of M1 and M2 markers in primary bone-marrow-derived macrophage (BMDM) cells. (K) Bar plots showing the messenger RNA (mRNA) expression levels of interleukin-6 (IL-6) and IL-10 in LPS-stimulated macrophages after LEV treatment. (L) Enzyme-linked immunosorbent assay (ELISA) results showing the secretion levels of IL-6 and IL-10 from macrophages under different treatment conditions. (M) Bar plot showing the effect of LEV treatment on FAO activity in macrophages under control and LPS-stimulated conditions. (N) Western blot analysis of glycolysis-related proteins (pyruvate kinase M2 [PKM2], glucose transporter 1 [GLUT1], and lactate dehydrogenase A [LDHA]) in macrophages. (O) Western blot analysis showing the knockdown efficiency of CPT1A using 3 different small interfering RNAs (siRNAs). (P) Western blot analysis showing the effect of CPT1A knockdown on the expression of macrophage polarization proteins after LEV treatment. (Q) Western blot analysis demonstrating the effect of the FAO inhibitor etomoxir on LEV-induced macrophage polarization protein expression. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 in the indicated groups.

Article Snippet: The recombinant mouse IL-10 (rmIL-10) protein was purchased from ABclonal Technology (no. RP01465).

Techniques: Derivative Assay, Micro-CT, Staining, Immunofluorescence, Labeling, Expressing, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Knockdown

Journal: Biomaterials Research

Article Title: Tryptophan-Enriched Lactobacillus rhamnosus GG-derived Nanovesicles Promote Alveolar Bone Regeneration through Macrophage Fatty Acid Oxidation

doi: 10.34133/bmr.0370

Figure Lengend Snippet: Assessment of interleukin-10 (IL-10) secretion from macrophages and in vitro osteogenic responses upon Lactobacillus rhamnosus GG-derived extracellular vesicle (LEV) treatment. (A) Histochemical staining for alkaline phosphatase (ALP; osteoblast marker) and tartrate-resistant acid phosphatase (TRAP; osteoclast marker) on paraffin sections of mouse maxillae from the periodontitis model. (B) A schematic diagram illustrating the in vitro co-culture systems (RAW264.7/MC3T3-E1 and THP-1/periodontal ligament stem cells [PDLSCs]) used to evaluate the paracrine pro-osteogenic effect of LEVs via macrophages. (C) ALP staining of MC3T3-E1 cells cultured in conditioned media (CM) derived from RAW264.7 macrophages treated with different conditions (NC, lipopolysaccharide [LPS], LEV, and LPS + LEV). (D) Alizarin Red S (ARS) staining of MC3T3-E1 cells cultured in CM derived from RAW264.7 macrophages treated with different conditions (NC, LPS, LEV, and LPS + LEV) to evaluate mineralized nodule formation. (E) Quantitative polymerase chain reaction (qPCR) analysis to detect the gene expression levels of osteogenic markers ( Runx2 , Osx , Bsp , and Ocn ) in MC3T3-E1 cells cultured in CM derived from RAW264.7 macrophages treated with different conditions (NC, LPS, LEV, LPS + LEV, siCPT1A, and αIL10). (F) Western blot analysis to detect the protein expression levels of osteogenic markers in MC3T3-E1 cells cultured in CM derived from RAW264.7 macrophages treated with different conditions (NC, LPS, LEV, LPS + LEV, siCPT1A, and αIL10). (G) ALP staining of PDLSCs cultured in CM derived from THP-1 macrophages treated with different conditions (NC, LPS, LEV, and LPS + LEV). (H) ARS staining of PDLSCs cultured in CM derived from THP-1 macrophages treated with different conditions (NC, LPS, LEV, and LPS + LEV) to evaluate mineralized nodule formation. (I) qPCR analysis to detect the gene expression levels of osteogenic markers ( RUNX2 , OSX , BSP , and OCN ) in PDLSCs cultured in CM derived from THP-1 macrophages treated with different conditions (NC, LPS, LEV, and LPS + LEV). (J) Western blot analysis to detect the protein expression levels of osteogenic markers in PDLSCs cultured in CM derived from THP-1 macrophages treated with different conditions (NC, LPS, LEV, and LPS + LEV). * P < 0.05 and *** P < 0.001 in the indicated groups.

Article Snippet: The recombinant mouse IL-10 (rmIL-10) protein was purchased from ABclonal Technology (no. RP01465).

Techniques: In Vitro, Derivative Assay, Staining, Marker, Co-Culture Assay, Cell Culture, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Expressing

Journal: Biomaterials Research

Article Title: Tryptophan-Enriched Lactobacillus rhamnosus GG-derived Nanovesicles Promote Alveolar Bone Regeneration through Macrophage Fatty Acid Oxidation

doi: 10.34133/bmr.0370

Figure Lengend Snippet: Measurement of the l -tryptophan–kynurenine–aryl hydrocarbon receptor (AhR) axis and carnitine palmitoyltransferase 1A (CPT1A) expression levels under Lactobacillus rhamnosus GG-derived extracellular vesicle (LEV) treatment. (A) Principal component analysis plot from tryptophan-targeted metabolomic analysis, showing the metabolic profile differences between LEV-treated macrophages and the control group. (B) A heatmap showing the levels of l -tryptophan and its downstream metabolite l -kynurenine with statistical in LEV-treated macrophages. (C) Single-sample gene set enrichment analysis (ssGSEA) analysis plot showing the gene set enrichment of the l -tryptophan catabolic process to the kynurenine pathway and the tryptophan transport pathway in LEV-treated macrophages. (D) Quantitative polymerase chain reaction (qPCR) analysis to detect the messenger RNA (mRNA) expression level of the key tryptophan metabolic enzyme indoleamine 2,3-dioxygenase 1 (IDO1) in macrophages treated with LEVs and l -tryptophan, with or without lipopolysaccharide (LPS)-induced inflammation. (E) qPCR analysis to detect the mRNA expression of M1 (CD86) and M2 (CD206) markers in macrophages treated with l -kynurenine. (F) Enzyme-linked immunosorbent assay (ELISA) analysis to detect the protein levels of IL-10 in macrophages treated with l -kynurenine, with or without LPS-induced inflammation. (G) Western blot analysis to detect the effect of the AhR-specific inhibitor CH223191 on l -kynurenine-induced CPT1A protein expression in macrophages. (H) RNA sequencing (RNA-seq) analysis showing the gene expression level of AhR in LEV-treated macrophages. (I) qPCR analysis to detect the mRNA expression levels of AhR in macrophages treated with LEV and LPS + LEV. (J) Western blot analysis to evaluate the effect of the vesicular endocytosis inhibitor Dynasore on LEV-induced intracellular AhR expression in the LPS inflammatory environment. (K) Western blot analysis to detect the effect of the AhR inhibitor CH223191 on LEV-induced CPT1A, CD163, and interleukin-10 (IL-10) protein expression. (L) ELISA analysis to detect the effect of the AhR inhibitor CH223191 on the secretion levels of interleukin-6 (IL-6) and IL-10 in LEV-treated macrophages. (M) Western blot analysis to detect the protein expression levels of nuclear AhR in LEV-treated macrophages. (N) Immunofluorescence images showing the subcellular localization of AhR in macrophages after treatment with the control, LPS, LEV, LPS + LEV, and CH223191 + LEV groups. (O) Coimmunoprecipitation (Co-IP) analysis to detect the effect of the AHR inhibitor CH223191 on the interaction between CPT1A and the 26S proteasome. * P < 0.05, *** P < 0.001, and **** P < 0.0001 in the indicated groups.

Article Snippet: The recombinant mouse IL-10 (rmIL-10) protein was purchased from ABclonal Technology (no. RP01465).

Techniques: Expressing, Derivative Assay, Metabolomic, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, RNA Sequencing, Gene Expression, Immunofluorescence, Co-Immunoprecipitation Assay

Journal: Biomaterials Research

Article Title: Tryptophan-Enriched Lactobacillus rhamnosus GG-derived Nanovesicles Promote Alveolar Bone Regeneration through Macrophage Fatty Acid Oxidation

doi: 10.34133/bmr.0370

Figure Lengend Snippet: Investigation of the aryl hydrocarbon receptor (AhR)/NAD(P)H:quinone oxidoreductase 1 (NQO1) signaling pathway and carnitine palmitoyltransferase 1A (CPT1A) protein stability in Lactobacillus rhamnosus GG-derived extracellular vesicle (LEV)-treated macrophages. (A) Venn diagram showing the intersection of differentially expressed genes (DEGs) from 3 datasets: macrophages from lipopolysaccharide (LPS)-treated AhR knockout mice, small interfering RNA (siRNA)-AhR-treated macrophages, and LEV-treated macrophages, used for screening downstream target genes of AhR. (B and C) Coimmunoprecipitation (Co-IP) analysis to detect the physical interaction between 3×Flag-CPT1A and S100A8 or NQO1. (D) Immunofluorescence staining of NQO1 and F4/80 in periodontal tissue, used to evaluate the effect of LEV intervention on NQO1 expression in macrophages. (E) Cellular immunofluorescence staining to observe the colocalization of NQO1 and CPT1A in macrophages after LEV stimulation. (F) Quantitative polymerase chain reaction (qPCR) analysis to detect the effect of LEVs on NQO1 messenger RNA (mRNA) expression levels in macrophages with or without LPS-induced inflammation. (G) Western blot analysis to detect the effect of LEVs on NQO1 protein expression levels in macrophages with or without LPS-induced inflammation. (H) Co-IP analysis to investigate the effect of LEVs on the interaction between NQO1 and CPT1A in an LPS inflammatory environment. (I) Chromatin immunoprecipitation (ChIP)–qPCR analysis to detect the effect of LEVs on the recruitment of AhR to the NQO1 promoter. (J) Assay for transposase-accessible chromatin using sequencing (ATAC-seq) traces of the Nqo1 gene locus in wild-type and unstimulated or LPS-stimulated bone-marrow-derived macrophages (BMDMs). The AhR/Arnt binding motif (xenobiotic response element [XRE]) in this region of the mouse genome is shown in the lower trace. (K) Washout experiment demonstrating the rescue effect of the AhR inhibitor CH223191 on LEV-mediated regulation of NQO1 protein expression. (L) Co-IP analysis to evaluate the effect of LEV treatment on the formation of a complex between CPT1A, NQO1, and the 26S proteasome. (M) Co-IP analysis to evaluate the binding of NQO1 and CPT1A to the 20S proteasome, serving as a negative control. (N) Co-IP analysis to investigate the role of NQO1 and its coenzyme nicotinamide adenine dinucleotide + hydrogen (NADH; 100 μM for 4 h) in regulating the interaction between CPT1A and the 26S proteasome. Cells were also treated with dicumarol (400 μM) for 4 h. (O) Western blot analysis to evaluate the knockdown efficiency of 2 NQO1 siRNAs. (P) Western blot analysis to evaluate the effect of NQO1 knockdown on the expression of M2 polarization-related proteins (CD163, arginase-1 [ARG1], and interleukin-10 [IL-10]) in macrophages. (Q) Cellular immunofluorescence staining to observe the effect of the NQO1 inhibitor dicumarol on LEV-induced M2 polarization and NQO1 expression. (R) Enzyme-linked immunosorbent assay (ELISA) analysis to evaluate the effect of NQO1 knockdown on LEVs’ regulation of interleukin-6 (IL-6) and IL-10 secretion in macrophages in an inflammatory environment. (S) Fatty acid oxidation (FAO) activity analysis to investigate the roles of AhR and NQO1 in LEV-induced macrophage metabolic reprogramming, showing the effects of 5 treatment groups (LEV, LEV + CH223191 , LEV + siNQO1, LEV + NADH, and LEV + siNQO1 + NADH). * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001 in the indicated groups.

Article Snippet: The recombinant mouse IL-10 (rmIL-10) protein was purchased from ABclonal Technology (no. RP01465).

Techniques: Derivative Assay, Knock-Out, Small Interfering RNA, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, ChIP-qPCR, Sequencing, Binding Assay, Negative Control, Knockdown, Enzyme-linked Immunosorbent Assay, Activity Assay