Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: The differential activation of CD14 and CD16 monocytes responding to endothelial and epithelial dysfunction. The profile scores of molecules mediating the interplays of classic monocytes with vascular endothelial cells (A-B) and non-classic monocytes with alveolar epithelial cells (C—D). E. Immunostaining for CDH5 and γH2AX in coronary artery cryo-section between KD patient and healthy donor, indicating endothelial injuries in KD. F. Expression level of CCR1 , DYSF , SELL , LMNB1, and XAF1 in CD14 classical monocytes among five groups. G. UMAP projection of CCR1 , DYSF , SELL , LMNB1, and XAF1 positive cells, respectively . H. UMAP projection of CCR1 , SELL and XAF1 triple positive monocytes among five groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Wilcoxon rank-sum test, adjusted for Bonferroni post hoc test, had been applied.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Activation Assay, Immunostaining, Expressing

Journal: Biochimica et Biophysica Acta. Molecular Basis of Disease

Article Title: Single cell RNA-seq resolution revealed CCR1 + /SELL + /XAF + CD14 monocytes mediated vascular endothelial cell injuries in Kawasaki disease and COVID-19

doi: 10.1016/j.bbadis.2023.166707

Figure Lengend Snippet: SELL+/CCR1+/XAF1+ CD14 monocytes enhanced the adhesion and damages to endothelial cells. A. Flow cytometry identified the higher percentages and median fluorescence intensity of SELL, CCR1, and LMNB1 in KD and COV. B. Immunostaining for CD14 and DYSF, XAF1 in isolated PBMCs. C. The ratio of DYSF positive CD14 monocytes in total classic monocytes among KD, COV, FLU, and healthy donors. And the median fluorescence intensity of XAF1 in CD14 monocytes among KD, FLU, and healthy donors. D-E. THP-1 had been stained with Calcein AM and transfected siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 before co-culture with HUVECs. Then the numbers of adhesion THP-1 with HUVECs were counted by every 20× field view. F. The expressions of TNFa and IL6 in HUVECs, and the ratio of γH2AX + cells in HUVECs after co-cultured with THP-1, which had been transfected with siRNAs of CCR1 , DYSF , SELL , LMNB1 , and XAF1 . G. The inhibition of SELL , CCR1 and XAF1 together in THP-1 significantly reduced the adhesion between monocytes and endothelial cells. H. Collaborated inhibiton of SELL , CCR1 and XAF1 in THP-1 decreased the expression of IL6 and TNF-α in HUVECs with lower ratio of rH2AX+ cells after co-culture with THP-1. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way analysis of variance with Bonferroni post hoc test was performed to analyze data. Bar, 100 μm.

Article Snippet: Cell smears fixated in 4 % PFA were blocked in blocking buffer (PBS containing 1 % BSA, 0.1 % Triton X-100, 5 % goat serum) for 1 h at room temperature, followed by incubation with rabbit anti-Dysferlin (1:200, Abcam, cat # ab124684), rabbit anti-XAF1 (1:200, Abcam cat #ab2254) antibodies and mouse anti-CD14 (1:200, Abcam cat #ab181470) diluted in PBS containing 1 % BSA, 5 % goat serum at 4 °C overnight.

Techniques: Flow Cytometry, Fluorescence, Immunostaining, Isolation, Staining, Transfection, Co-Culture Assay, Cell Culture, Inhibition, Expressing

Journal: Scientific reports

Article Title: Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins.

doi: 10.1038/s41598-022-08387-7

Figure Lengend Snippet: Figure 2. Analysis of soluble CD14 (sCD14) levels in haemodialysis (HD) and non-CKD controls (control). (A) Soluble CD14 levels. The number of samples (n) that each analyte was detected in is noted for each group. Data presented at median and interquartile range. Statistical significance, ***p < 0.001, ****p < 0.0001. (B) Spearman’s correlation between sCD14 and trimethylamine N-oxide (TMAO) levels.

Article Snippet: The S100B ELISA kit was performed in accordance with manufacturer guidelines, with the alteration that samples were undiluted. sCD14 levels in HD patients was previously recorded and reported87, for measurements in non-CKD controls the same CD14 ELISA kit (DC140, R&D Systems, UK) was used, and run in accordance with manufacturer guidelines.

Techniques: Control

Journal: Scientific reports

Article Title: Blood-brain barrier and gut barrier dysfunction in chronic kidney disease with a focus on circulating biomarkers and tight junction proteins.

doi: 10.1038/s41598-022-08387-7

Figure Lengend Snippet: Figure 1. Analysis of serum biomarkers in haemodialysis (HD) and non-CKD controls (control). (A) Brain-derived neurotrophic factor (BDNF), (B) Neuron-specific enolase (NSE) were measured by enzyme- linked immunosorbent assay (ELISA). (C) Trimethylamine N-oxide (TMAO) was measured by liquid chromatography-mass spectrometry (LC–MS). The number of samples (n) that each analyte was detected in is noted for each group. Data presented at median and interquartile range. Statistical significance, ***p < 0.001, ****p < 0.0001. (D) Spearman’s correlation between BDNF and TMAO levels. (E) Spearman’s correlation between NSE and TMAO levels.

Article Snippet: The S100B ELISA kit was performed in accordance with manufacturer guidelines, with the alteration that samples were undiluted. sCD14 levels in HD patients was previously recorded and reported87, for measurements in non-CKD controls the same CD14 ELISA kit (DC140, R&D Systems, UK) was used, and run in accordance with manufacturer guidelines.

Techniques: Control, Derivative Assay, Enzyme-linked Immunosorbent Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Nature Communications

Article Title: Androgens predispose males to monocyte-mediated immunopathology by inducing the expression of leukocyte recruitment factor CXCL1

doi: 10.1038/s41467-020-17260-y

Figure Lengend Snippet: a Gating strategy to identify monocyte subsets in healthy human blood donors. b Percentage of classical (CD14 + CD16 − ), intermediate (CD14 + CD16 + ), and non-classical (CD14 − CD16 + ) monocytes in the peripheral blood of healthy men and women ( n m/f CD14+CD16− = 24/25; n m/f CD14+CD16+ = 24/25; n m/f CD14−CD16+ = 24/25; pooled data from samples collected over a time frame of nine months; depicted are the means). Histogram and graphs showing the MFI of c CD86, d CCR2, and e CX 3 CR1 on peripheral monocyte subpopulations from healthy male and female human blood donors ( c n m/f CD14+CD16− = 8; n m/f CD14+CD16+ = 8; n m/f CD14−CD16+ = 8; d n m/f CD14+CD16− = 14; n m/f CD14+CD16+ = 14; n m/f CD14−CD16+ = 14; e n m/f CD14+CD16− = 14; n m/f CD14+CD16+ = 14; n m/f CD14−CD16+ = 14; pooled data of samples collected over a time frame of nine months; Depicted are the means). f Transcriptome analysis of CD14 + monocytes from men and women. PBMCs from male ( n = 4) and female ( n = 3) healthy donors (25–49 years of age) were stimulated with LPS, and CD14 + monocytes were isolated using MACS. RNA was isolated, transcribed into cDNA, and subjected to RNA Seq analysis. Venn diagram showing differentially regulated genes from men and women following LPS stimulation compared with the corresponding medium control (log2FoldChange > 2 and a padj value < 0.05). g PANTHER GO-slim analysis of biological processes, and h FDR- q values of GO terms connected to leukocyte migration in men and women conducted with GOrilla analysis(Table ). P -values were calculated using two-tailed paired analysis; Wilcoxon matched-paired signed rank test ( d , h ), * P < 0.05; **P < 0.01. Source data are provided as a Source Data file.

Article Snippet: CD14 + monocytes were isolated with the anti-Human CD14 Magnetic Particles Kit (Becton Dickinson) according to the instructions of the manufacturer.

Techniques: Isolation, RNA Sequencing, Control, Migration, Two Tailed Test

Journal: Nature Communications

Article Title: Androgens predispose males to monocyte-mediated immunopathology by inducing the expression of leukocyte recruitment factor CXCL1

doi: 10.1038/s41467-020-17260-y

Figure Lengend Snippet: a Gating strategy and representative dot plots showing cytokines produced by classical monocytes upon stimulation. PBMCs from men and women were stimulated for 6 h with E. histolytica lysate (0.1 mg/mL) or LPS (0.1 µg/mL), and intracellular expression of b TNF, c CCL2, and d CXCL1 by classical monocytes was measured by flow cytometry and compared to unstimulated controls ( b n m/f w/o = 10/10; n m/f E. his = 13/14; n m/f LPS = 13/14; c n m/f w/o = 10/10; n m/f E. his = 13/14; and n m/f LPS = 13/14; d n m/f w/o = 10/10; n m/f E. his = 13/14; n m/f LPS = 13/14; pooled data from samples collected over a time frame of nine months; depicted are the means). Hierarchical Stochastic Neighbor Embedding (HSNE) analysis of e E. histolytica- CD14 + CD16 − and f LPS-stimulated CD14 + CD16 − classical monocytes. Cluster (1), TNF + CXCL1 − CCL2 − ; cluster (2), TNF + CXCL1 + CCL2 − ; and cluster (3), TNF + CXCL1 + CCL2 + (calculated from classical CD14 + monocytes) (Table ). P -values were calculated using two-tailed grouped analysis: Mann–Whitney test ( b , d ), ** P < 0.01. Source data are provided as a Source Data file.

Article Snippet: CD14 + monocytes were isolated with the anti-Human CD14 Magnetic Particles Kit (Becton Dickinson) according to the instructions of the manufacturer.

Techniques: Produced, Expressing, Flow Cytometry, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Androgens predispose males to monocyte-mediated immunopathology by inducing the expression of leukocyte recruitment factor CXCL1

doi: 10.1038/s41467-020-17260-y

Figure Lengend Snippet: MACS-purified CD14 + monocytes from men were stimulated for 24 h with LPS (0.1 µg/mL). The concentration of a – c CXCL1 and d – f CCL2 in the supernatant was examined as follows: a , d in the presence of an αTNF mAb or an isotype IgG mAb control ( n w/o; Isotype ctrl; αTNF = 3); b , e in the presence of αTNFRI (1 µg/mL), an αTNFRII mAb (10 µg/mL), or an isotype control IgG mAb ( n w/o; Isotype ctrl; αTNFRI; αTNFRII; αTNFRI+II = 4); c , f in the presence of inhibitors of DNA transcription (4 µM actinomycin D), p38 MAPK (10 µM SB202190), PI3K (10 µM LY294002), NF-κB (5 µM BAY11-7085), of AP-1 (17 µM Tanshinone) and of JNK (10 µM SP600125) ( c n w/o stimulation; w/o; Transcription; p38 MAPK; PI3K; NFκB; AP-1; JNK = 3; f n w/o stimulation; w/o; Transcription; p38 MAPK; PI3K; NFκB; AP-1; JNK = 3). The data shown are one representative out of three independent experiments. a – f Depicted are the means. g Proposed scheme outlining differences in the CCL2 and CXCL1 expression by LPS-stimulated CD14 + monocytes, based on the study by Lo et al. , Cai et al. and Koryankina et al. (green line = part of the signal transduction for CCL2 and CXCL1, blue interrupted line=inhibition of CXCL1 expression, yellow line=induces CCL2, black line = induced by androgens, and gray dotted line = interaction assumed). P -values were calculated using two-tailed grouped analysis using an ordinary one-way ANOVA with Bonferoni Correction ( a – f ), * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ( a F (degree of freedom (DFd) (12, 6)) = 273, 9; b F (4, 15) = 300, 3; c F (7, 21) = 48, 39; d F (2, 6) = 14, 10; e F (4, 14) = 40, 86; f F (7, 16) = 93,23). Source data are provided as a Source Data file.

Article Snippet: CD14 + monocytes were isolated with the anti-Human CD14 Magnetic Particles Kit (Becton Dickinson) according to the instructions of the manufacturer.

Techniques: Purification, Concentration Assay, Control, Expressing, Transduction, Inhibition, Two Tailed Test