Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: ( a ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression by 24-h control, IFN-γ- and/or TNF-α-stimulated undifferentiated KCs or HPV16+KCs. Gene expression was normalized using GAPDH as the calibrator gene. Fold changes over control-stimulated undifferentiated KCs were calculated and depicted. ( b ) Enzyme-linked immunosorbent assay for CCL2, RANTES, IL-8 and CXCL9 in cleared supernatants of 24-h control, IFN-γ- and/or TNF-α-stimulated undifferentiated KCs or HPV16+KCs. ( c ) PBMCs migration towards cleared supernatants of 24-h control, IFN-γ- and/or TNF-α-stimulated KCs or HPV16+KCs. A representative example of three different donors is shown. ( d ) RelA phosphorylation, acetylation and total levels in KCs and HPV16+KCs stimulated with TNF-α for 0, 5, 15 and 30 min. ( e ) RelA acetylation and total levels at steady state in three human primary KC donor pools originating from human foreskin keratinocytes (HFKs), human vaginal keratinocytes (HVKs) or human cervical keratinocytes (HCKs) and two HPV16+genome-transfected primary KC pools of foreskin (HFK16) or vaginal (HVK16) origin. ( f ) RT–qPCR of CCL2, RANTES, IL-8 and CXCL9 in HPV16+KCs and KCs. Gene expression was normalized using GAPDH as the calibrator gene. Gene expression in HPV16+KCs was standardized over KCs. All data are representative for at least three independent experiments. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Control, Gene Expression, Enzyme-linked Immunosorbent Assay, Migration, Phospho-proteomics, Transfection

Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: Microarray intensities for ( a ) all KATs , HDACs and SIRTs , and ( b ) IFRD1 in four independent KCs and four independent hrHPV+KCs represented in a box plot. The box contains the 1st quartile up to the 3rd quartile; the median is represented as a line; whiskers represent the values of the outer two quartiles. ( c ) IFRD1 mRNA expression of one representative control primary KC culture and two HPV16+KC cultures (left panel), in HFK16 cells transfected with siControl or siHPV16 (middle panel) and in primary KCs that are either mock infected or infected with native HPV16 virions (right panel), as measured by RT–qPCR. ( d ) IFRD1 protein expression in three human primary keratinocyte (KC) donor pools originating from human foreskin keratinocytes (HFKs), human vaginal keratinocytes (HVKs) or human cervical keratinocytes (HCKs) and two HPV16+genome-transfected primary KC pools of foreskin (HFK16) or vaginal (HVK16) origin (left panel) in HFK16 cells transfected with siControl or siHPV16 (middle panel) and in primary KCs that are either mock infected or infected with native HPV16 virions (right panel), as measured by western blot. ( e ) Immunohistochemical staining for IFRD1, HPV16 E2, p16 and negative antibody control of a vulvar intraepithelial neoplasia (VIN) lesion, one representative donor of two shown. Counterstaining was done using haematoxylin. Scale bar, 500 μm. ( f ) IFRD1, RelA K310 acetylation and total RelA levels in 24-h non- or IFN-γ- and TNF-α-stimulated control or IFRD1 knockdown (KD) HPV16+KCs. ( g ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in steady-state control or IFRD1 KD HPV16+KCs. ( h ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and/or TNF-α-stimulated control or IFRD1 KD HPV16+KCs. ( i ) Enzyme-linked immunosorbent assay for CCL2, RANTES, IL-8 and CXCL9 in cleared supernatants of 24-h non- or IFN-γ- and/or TNF-α-stimulated control or IFRD1 KD HPV16+KCs. ( j ) PBMCs migration towards cleared supernatants of 24-h non- or IFN-γ- and TNF-α-stimulated control or IFRD1 KD HPV16+KCs. A representative example of three different donors is shown. These data are representative for at least three independent experiments. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Microarray, Expressing, Control, Transfection, Infection, Quantitative RT-PCR, Western Blot, Immunohistochemical staining, Staining, Knockdown, Enzyme-linked Immunosorbent Assay, Migration

Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: ( a ) Microarray intensities for EGFR in KCs ( n =4) and hrHPV+KCs ( n =4) represented in a box plot. ( b ) Histogram of EGFR surface protein expression on KCs and HPV16+KCs, as determined by flow cytometry. ( c ) RT–qPCR of EGFR expression in KCs transfected with complementary DNA for E2, E5, E1+E2+E6+E7 or empty control. ( d ) RT–qPCR of IFRD1 expression in KCs and HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml −1 anti-EGFR or anti-CD20. ( e ) IFRD1, RelA K310 acetylation and total RelA levels in KCs and HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml −1 anti-EGFR or anti-CD20. ( f ) Quantified protein levels of IFRD1, RelA K310 acetylation and RelA over β-actin in HPV16+KCs treated for 72 h with 0, 0.1, 1 or 10 μg ml −1 anti-EGFR (two-dimensional western blot). The expression levels of the 0 μg ml −1 -treated HPV+KCs were set as 100%. ( g ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated, anti-CD20- or anti-EGFR-treated HPV16+KCs (left) and KCs (right). ( h ) Enzyme-linked immunosorbent assay for CCL2, RANTES, IL-8 and CXCL9 in cleared supernatants of 24-h non- or IFN-γ- and TNF-α-stimulated, anti-CD20- or anti-EGFR-treated HPV16+KCs (left) and KCs (right). ( i ) RT–qPCR of IFRD1 expression in HPV16+KCs treated with inhibitors of PI3K (LY94002, 25 μM), mTOR (rapamycin, 50 nM), MEK1 (PD98059, 50 μM), RAF (GW5074, 20 μM) and JNK (SP60025, 20 μM). Gene expression was normalized using GAPDH as the calibrator gene. Fold changes over control were calculated and depicted. These data are representative for at least three independent experiments, except for h that was performed once. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Microarray, Expressing, Flow Cytometry, Quantitative RT-PCR, Transfection, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Gene Expression

Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: ( a ) RelA K310 acetylation and total RelA levels in KCs and HPV16+KCs treated with decreasing doses of entinostat (40, 20, 10 and 2 μM), SAHA (10, 5 and 1 μM), TSA (5, 1 and 0.333 μM) or NaBu (10, 5 and 1 mM). RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in steady state ( b ) or 24-h non- or IFN-γ- and TNF-α-stimulated ( c ) control or entinostat (10 μM) pre-treated HPV16+KCs. ( d ) Total RelA levels and RelA K310 acetylation in non- or entinostat-treated control or RelA knockdown (KD) HPV16+KCs. ( e ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated non- or entinostat-treated control or RelA knockdown (KD) HPV16+KCs. ( f ) RT–qPCR of EGFR expression in KCs and HPV16+KCs treated with increasing doses of entinostat (0, 10 or 40 μM). Gene expression was normalized using GAPDH as the calibrator gene. ( g ) IFRD1 in control or entinostat (10 μM) pre-treated HPV16+KCs. ( h ) RT–qPCR of IFRD1 and EGFR expression in control or IFRD1 KD HPV16+KCs. Gene expression was normalized using GAPDH as the calibrator gene. Histogram ( i ) and geomean ( j ) of EGFR expression on control or IFRD1 KD HPV16+KCs, as determined by flow cytometry. s.e.m. of two independent experiments. These data are representative for at least two independent experiments. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Control, Knockdown, Gene Expression, Flow Cytometry

Journal: Nature Communications

Article Title: The interferon-related developmental regulator 1 is used by human papillomavirus to suppress NFκB activation

doi: 10.1038/ncomms7537

Figure Lengend Snippet: ( a ) IFRD1, RelA acetylation and total RelA levels at steady state in three KC donors and three HPV16-induced CxCa lines. ( b ) RT–qPCR of IFRD1 , CCL2 , RANTES , IL-8 and CXCL9 expression, and IFRD1 protein levels in steady-state control or IFRD1 KD Caski cells. ( c ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated control or IFRD1 KD Caski cells. ( d ) Histogram of EGFR expression on three HPV16-induced CxCa lines. ( e ) Geomean of EGFR expression on KCs and CxCa, as determined by flow cytometry. s.e.m. of two independent experiments. ( f ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated anti-CD20- or anti-EGFR-treated Caski cells. ( g ) IFRD1 and RelA K310 acetylation status in Caski cells treated for 72 h with 0, 1 or 10 μg ml −1 anti-EGFR (cetuximab) or anti-CD20 (rituximab). ( h ) RT–qPCR of IFRD1 expression in KCs and Caski cells treated for 72 h with 0, 0.1, 1 or 10 μg ml −1 anti-EGFR or anti-CD20. ( i ) RT–qPCR of CCL2 , RANTES , IL-8 and CXCL9 expression in 24-h non- or IFN-γ- and TNF-α-stimulated control (dimethylsulphoxide (DMSO)) or entinostat-treated Caski cells. ( j ) Schematic representation of IFRD1-mediated RelA (de-)acetylation. (I) In KCs, RelA acetylation is positively regulated by KATs, resulting in the production of pro-inflammatory cytokines. HDACs may suppress this process. (II) In HPV+KCs, elevated EGFR levels can induce the expression of IFRD1, which can mediate RelA deacetylation by forming a bridge between RelA and HDAC1 and/or -3, hampering pro-inflammatory gene expression. (III) Interfering with EGFR signalling (1 and 2) or HDAC function (3) may lower IFRD1 levels, restoring the RelA acetylation balance, augmenting pro-inflammatory gene expression. Error bars indicate s.d. P values were determined using Welch-corrected unpaired t -tests. * P <0.05, ** P <0.01 and *** P <0.001.

Article Snippet: Immunodetection was achieved with anti-p65 (1:1,000, sc-372, Santa Cruz), anti-phospho-p65 (Ser536; 1:1,000, #3033 Cell Signaling Technology (CST)), anti-acetyl-p65 (Lys310; 1:1,000, #3045 CST), anti-IFRD1 (1:400, T2576 Sigma-Aldrich), β-actin (1:10,000, Sigma-Aldrich) primary antibodies, and horseradish peroxidase-coupled anti-mouse (1:5,000; CST) and horseradish peroxidase-coupled anti-rabbit (1:5,000, CST) secondary antibodies.

Techniques: Quantitative RT-PCR, Expressing, Control, Flow Cytometry, Gene Expression

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 1. Clusterin inhibits tumor growth and angiogenesis. A, growth of subcutaneous tumors derived from RasMyc cells transduced with a retrovirus expressing murine clusterin and/or the puromycin resistance gene. Inset, immunoblot of clusterin levels in Ras and RasMyc colonocytes. B, growth of subcutaneous tumors derived from HCT116 cells transduced with an empty vector or the retrovirus expressing human clusterin. Regression lines represent average rates of growth. The P value refers to the difference in regression coefficients. Inset, immunoblot of clusterin levels in transduced cells. C, in vivo bioluminescent imaging of representative animals from the experiment in B. Mice were photographed at days 3 and 21 after injection. No less than five animals per group were used in all these experiments. D, immunohistochemical staining of Ki-Ras and Ki-Ras/Myc tumors with an anti-clusterin antibody. Cytoplasmic staining for clusterin is depicted in brown whereas nuclei are counterstained in blue. E, alignment of the second TSR of murine thrombospondin-1 with the C9 protein and clusterin. F, hemoglobin content of Matrigel pellets 7 d after injection. Matrigels were admixed with p53-null mouse colonocytes transduced with either empty vector (blue bar) or clusterin retrovirus (red bar) and injected s.c. into syngeneic host animals. G, H&E staining of RasMyc/vector (top) and RasMyc/clusterin (bottom) tumor sections. Perfused blood vessels contain numerous RBC, which could be clearly seen under higher magnification (inset). The scatter plot represents densities of perfused blood vessels in four individual tumors. H, immunohistochemical staining of control (top) and clusterin-overexpressing (bottom) HCT116 tumor sections with antibody recognizing the endothelial cell surface antigen CD31. The scatter plot represents the densities of CD31-positive blood vessels in four individual tumors.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Derivative Assay, Transduction, Expressing, Western Blot, Plasmid Preparation, In Vivo, Imaging, Injection, Immunohistochemical staining, Staining, Control

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 2. Clusterin is regulated by miR-17~92 via the TGFβ pathway. A, immunoblotting analysis of clusterin expression levels in the following cell lines. Left, Ras-only mouse colonocytes transduced with either empty vector (Ras/Puro) or the miR-17~92–encoding retrovirus (Ras/miR-17~92). Right, RasMyc cells transfected with scrambled or anti–miR-17~92 2′-O-methyl oligoribonucleotides. B, changes in expression levels of thrombospondin-1 (THBS1) and clusterin mRNAs in HCT116 Dicerhypo (left) and A172 (right) cells after transfection with the indicated microRNA mimics. mRNA levels in HCT116 and A172 cells were profiled using Affymetrix microarrays and qPCR as described in Materials and Methods. C, activation of TGFβ signaling in Ras colonocytes. Ras cells treated with vehicle or 10 ng/mL of TGFβ1 for 30 min were analyzed by immunoblotting for phosphorylated Smad3 (pSmad3) and total Smad3. D, measurement of TGFβ effects on TSR proteins. Ras cells were treated with increasing doses of TGFβ1 for 48 h and lysates were immunoblotted for clusterin and CTGF proteins. Tsp-1 was detected in conditioned medium. E, immunoblotting analysis of TSR proteins in Ras/vector, Ras/miR-17~92 or c-Myc cells cultured in the absence or presence of TGFβ1 (5 ng/mL) for 48 h. F, CLU mRNA levels in Ras/vector and Ras/17~92 cells before and after (24 h) stimulation with TGFβ, as measured by qPCR. Expression levels of CLU are adjusted to those of glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Western Blot, Expressing, Transduction, Plasmid Preparation, Transfection, Activation Assay, Cell Culture

Journal: Cancer Research

Article Title: The Myc–miR-17∼92 Axis Blunts TGFβ Signaling and Production of Multiple TGFβ-Dependent Antiangiogenic Factors

doi: 10.1158/0008-5472.can-10-2412

Figure Lengend Snippet: Figure 3. miR-17~92 targets endogenous TGFβ receptor II. A, luciferase sensor assay. Constructs tested were psiCHECK-2 derivatives containing a single miR-17/20a binding site from TGFBR2 3′-UTR in either wild-type (wt) or seed-mutated (mut) conformation. Cells were additionally cotransfected with miR-17 or control mimic. Results are expressed as ratios of renilla to firefly luciferase, the latter being constitutively expressed from the same vector and serving as a control for transfection efficiency. Sequence alignment corresponds to positions 268 to 274 of TGFBR2 3′-UTR and mature hsa-miR-17. Arrows indicate mutated nucleotides. B, expression levels of TGFBR2 mRNA in DLD1 Dicerhypo cells 10 h after transfection with microRNA mimics (25 nmol/L). mRNA levels were quantified by microarray. C, immunoblotting analysis of TGFBR2 and clusterin expression levels in Ras cells transfected with 10 nmol/L of nontargeting siRNA pool or siRNA pool targeting mouse TGFBR2. D and E, immunoblotting analysis of TGFBR2 and Smad3 in Ras cells 20 h after transfection with microRNA mimics (25 nmol/L). Bottom, quantitations of Western blots above.

Article Snippet: For thrombospondin-1 expression is, either cell lysates or conditioned media were used. ranes were probed with antibodies to clusterin, , TGFBR2 (Santa Cruz Biotechnology and Abcam), 4 (Santa Cruz Biotechnology), Smad2 and Smad3 ogen), phosphorylated Smad3 (Cell Signaling), and (Ab-11, Lab Vision) according to the recommendaof the manufacturer.

Techniques: Luciferase, Construct, Binding Assay, Control, Plasmid Preparation, Transfection, Sequencing, Expressing, Microarray, Western Blot

Journal: Frontiers in Immunology

Article Title: Activated Neutrophils Secrete Chitinase-Like 1 and Attenuate Liver Inflammation by Inhibiting Pro-Inflammatory Macrophage Responses

doi: 10.3389/fimmu.2022.824385

Figure Lengend Snippet: Primer sequence.

Article Snippet: Recombinant mouse CHIL1 (2649-CH-050) was from R&D Systems (Minneapolis, MN).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Activated Neutrophils Secrete Chitinase-Like 1 and Attenuate Liver Inflammation by Inhibiting Pro-Inflammatory Macrophage Responses

doi: 10.3389/fimmu.2022.824385

Figure Lengend Snippet: Microarray analysis was performed in N-Neu and A-Neu. (A) Microarray heatmap data showing the expression level of transcript between N-Neu and A-Neu. The mRNA expressions were expressed as follows: Bright green, under expression; black, no change; bright red, overexpression. n=3. Row-based Z-score normalized. (B) GO enrichment analysis of differentially expressed genes in S1P activated neutrophils. (C) Venn diagram of gene expression overlap between GO terms including Regulation of Cell Communication, Cytokine Secretion, Inflammatory Response and Extracellular Space clusters. (D) Volcano plot of A-Neu gene expression compared with N-Neu. The threshold values were fold change≥|2| and P value ≤ 0.05. Red dots and blue dots represent up-regulated and down-regulated genes, respectively. Gray dots indicate genes with no statistically significant differences. The arrow indicated the location of Chil1. A-Neu, activated neutrophil; N-Neu, non-activated neutrophil; Il1b, Interleukin 1 beta; Chil1, Chitinase-like 1; Ccl3, Chemokine (C-C motif) ligand 3; Il1a, Interleukin 1 alpha; Tnf, tumor necrosis factor.

Article Snippet: Recombinant mouse CHIL1 (2649-CH-050) was from R&D Systems (Minneapolis, MN).

Techniques: Microarray, Expressing, Over Expression, Gene Expression

Journal: Frontiers in Immunology

Article Title: Activated Neutrophils Secrete Chitinase-Like 1 and Attenuate Liver Inflammation by Inhibiting Pro-Inflammatory Macrophage Responses

doi: 10.3389/fimmu.2022.824385

Figure Lengend Snippet: Chil1 was positively correlated with Ly6g in the injured liver of mice and could be secreted by A-Neu. (A) The mRNA expression of Chil1 was examined by RT-qPCR in the injured liver of MCDHF-treated mice in indicated times (n=6). (B) S1P stimulated neutrophils for 2 hours, then PBS washed the cells, and continued to culture for 6 hours, the secretion of CHIL1 in cell supernatant was detected by ELISA, n=4. (C) Correlation analysis between Chil1 mRNA expression with neutrophil marker Ly6g , macrophage marker Adgre1 , hepatocyte marker Alb and myofibroblast marker Acta2 in the injured liver of MCDHF-treated mice. Nonparametric test (Kruskal-Wallis test) was used in (A) Student’s t test was used in (B) Pearson’s test was used in (C) *P < 0.05 vs. MCDHF treated group for 0 days or control. MCDHF, methionine-choline-deficient and high-fat diet; S1P, Sphingosine 1-phosphate; Chil1, Chitinase-like 1; Ly6G, lymphocyte antigen 6 complex, locus G; Adgre1, adhesion G protein-coupled receptor E1; Acta2, actin alpha 2; Alb, albumin.

Article Snippet: Recombinant mouse CHIL1 (2649-CH-050) was from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Marker, Control

Journal: Frontiers in Immunology

Article Title: Activated Neutrophils Secrete Chitinase-Like 1 and Attenuate Liver Inflammation by Inhibiting Pro-Inflammatory Macrophage Responses

doi: 10.3389/fimmu.2022.824385

Figure Lengend Snippet: Recombinant CHIL1 inhibited the pro-inflammatory phenotype of macrophages. (A) Schematic representation of the experimental design: bone marrow-derived macrophages were stimulated with LPS 10 ng/mL for 3 hours, washed with PBS, and then treated with Vehicle (PBS) or rCHIL1 (100 ng/mL) for 24 hours respectively. (B) Macrophages were harvested and the mRNA expression levels of genes Ccl4 , Tnf and Nos2 were evaluated using RT-qPCR, presented relative to Gapdh , (control group, n=3; LPS group, n=6). The protein expressions of CCL4, TNF and NOS2 in liver were detected by CBA (C, D) and WB (E) n=4. The results were presented as mean ± SD. Two-way ANOVA was used. *P < 0.05. rCHIL1, Recombinant CHIL1; Ccl4, C-C chemokine motif ligand 4; Tnf, tumor necrosis factor; Nos2, nitric oxide synthase 2; LPS, lipopolysaccharide.

Article Snippet: Recombinant mouse CHIL1 (2649-CH-050) was from R&D Systems (Minneapolis, MN).

Techniques: Recombinant, Derivative Assay, Expressing, Quantitative RT-PCR, Control

Journal: Frontiers in Immunology

Article Title: Activated Neutrophils Secrete Chitinase-Like 1 and Attenuate Liver Inflammation by Inhibiting Pro-Inflammatory Macrophage Responses

doi: 10.3389/fimmu.2022.824385

Figure Lengend Snippet: Immunodepletion of CHIL1 in A-Neu supernatant attenuated the inhibitory effect on pro-inflammatory macrophages. (A) Schematic representation of the experimental design: bone marrow-derived macrophages were stimulated with LPS 10 ng/mL for 3 hours, washed with PBS, and then cultured in the presence of A-Neu supernatant (SUP) or A-Neu supernatant immunodepleted with anti-CHIL1 (SUP-anti-CHIL1). Expressions of CCL4, TNF and NOS2 in indicated groups were detected by RT-qPCR ( B , n=6), CBA ( C, D , n=4) and WB ( E , n=3). The results were presented as mean ± SD. One-way ANOVA was used. *P < 0.05. A-Neu, activated neutrophil; N-Neu, non-activated neutrophil; SUP, activated neutrophil supernatant; SUP-anti-CHIL1, activated neutrophil supernatant immunodepletion CHIL1; Ccl4, C-C chemokine motif ligand 4; Tnf, tumor necrosis factor; Nos2, nitric oxide synthase 2; LPS, lipopolysaccharide.

Article Snippet: Recombinant mouse CHIL1 (2649-CH-050) was from R&D Systems (Minneapolis, MN).

Techniques: Immunodepletion, Derivative Assay, Cell Culture, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: Activated Neutrophils Secrete Chitinase-Like 1 and Attenuate Liver Inflammation by Inhibiting Pro-Inflammatory Macrophage Responses

doi: 10.3389/fimmu.2022.824385

Figure Lengend Snippet: The schematic diagram of the main content of this study. A-Neu secrete CHIL1 and reduce liver inflammation by inhibiting the pro-inflammatory macrophage response. A-Neu, activated neutrophil; N-Neu, non-activated neutrophil; Ccl4, C-C chemokine motif ligand 4; Tnf, tumor necrosis factor; Nos2, nitric oxide synthase 2; S1P, Sphingosine 1-phosphate; Chil1, Chitinase-like 1.

Article Snippet: Recombinant mouse CHIL1 (2649-CH-050) was from R&D Systems (Minneapolis, MN).

Techniques: