Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: WT and CD5L − mice were subjected to cecal ligation and puncture (CLP) surgery to induce moderate disease severity. a Kaplan–Meier survival curves were generated to compare survival between the two groups and significance was determined by log-rank (Mantel-Cox) test. Graphical representation of pooled individuals ( n = 15 in each group) from 5 independent experiments. b Quantification of body weight loss in the experimental setting described in a . Data are presented as mean values ± SEM. c – e WT and CD5L − mice were subjected to mid-grade CLP; control mice (sham) underwent the same surgical procedure but without ligation and puncture of the cecum. Mice were sacrificed 6, 24, or 72 h after surgery. Pooled data from at least 2 independent experiments. c CFU counts of the indicated tissues from WT and CD5L − mice after mid-grade CLP. Statistical differences between groups were analyzed by two-tailed Mann-Whitney test. DL, detection limit = 20 CFU. Mice per group at 6, 24, and 72 h: Peritoneum WT: 7, 6, and 7; peritoneum CD5L − : 6, 6, and 8. Blood WT: 7, 6, and 7; blood CD5L − : 8, 6, and 8. Lung, liver, and kidney WT: 7, 7, and 7; lung, liver and kidney CD5L − : 7, 6, and 8. d Absolute number of total CD45 + leukocytes, CD45 + CD11b + Ly6G + neutrophils, CD45 + CD11b + CD11c - F4/80 + macrophages and Ly6C + and CD206 + subsets within macrophage population, assessed by flow cytometry of cell populations in the peritoneal cavity. Statistical comparisons were drawn after performing two-tailed unpaired t-tests with Welch’s correction. Mice per group at 6, 24, and 72 h: Sham, WT, and CD5L − : 4, 4, and 4. CLP WT: 7, 7, and 7; CLP CD5L − : 7, 6, and 8. Floating bars show the minimum, average (line), and maximum values within each group ( c-d ). e Fold change in the indicated cytokines between CD5L − and WT mice, quantified by bead-based multiplex immunoassay in samples from the peritoneal cavity (left panel) and blood serum (right panel).

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Ligation, Generated, Two Tailed Test, MANN-WHITNEY, Flow Cytometry, Multiplex Assay

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Principal component analysis (PCA) of RNA-seq expression data for the 4 groups of peritoneal cells [WT and CD5L − mice, either naive (0 h) or 6 h after CLP; n = 3]. b Volcano plot depicting differentially expressed genes in CD5L − vs. WT mice, 6 h after CLP. Red dots represent genes expressed at higher levels in CD5L − mice, while black dots represent genes with higher expression levels in WT controls. Grey dots represent genes bellow the cutoff of significant (|log 2 fold change| ≥ 0.5 and adjusted P values ≤ 0.05). Differential expression was evaluated using the Wald’s test, followed by adjustment for multiple testing with the Benjamini-Hochberg correction. Log 2 -fold change values were shrunk with the apeglm method to increase the signal-over-noise ratio of the effect size. c Dot plot of gene set enrichment analysis (mouse hallmark gene set collection) for CD5L − vs. WT mice, 6 h after CLP. The diameter of the dot indicates the degree of significance of the ontology term. Red dots represent terms enriched in CD5L − mice, while black dots represent terms enriched in WT mice. d Heatmap of the relative expression values (z-score of each gene across samples) of the top 7 upregulated pathways. Only differentially expressed genes were represented, excluding genes belonging to more than one pathway.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: RNA Sequencing Assay, Expressing

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Two-dimensional t-distributed stochastic neighbor embedding (t-SNE) visualization of cytokines, CFUs and neutrophil recruitment for a perplexity of 8. Each ellipse describes a phenotypic subtype and are derived from 36 different measurements (seven for WT, five for IP-treated, and six for the remaining groups). Measurements were log 2 transformed and normalized by the mean value of the “untreated” or the CD5L − subgroups for each setup [intravenous (IV); intraperitoneal (IP) or WT/CD5L − ]. b Linear discriminant analysis (LDA) biplot showing the overall profile of treatment of CLP with rCD5L administered IV or IP, and mid-grade CLP in WT or CD5L − mice. Ellipses show 95% confidence intervals for each treatment and vectors represent the contribution of each variable to the overall variance.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Derivative Assay, Transformation Assay

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Normalized counts of Cd5l transcripts obtained from RNA-seq analysis of peritoneal cells of WT mice at indicated times after CLP. Mice per group: 3. b RT-qPCR quantification of Cd5l expression, normalized with Hprt1 , in peritoneal cells and liver tissue at indicated times after CLP. Mice per group: 3. c ELISA quantification of CD5L in peritoneum and blood of WT mice at indicated times after mid-grade CLP, or in sham operated animals. Mice per group: at 0 h: 8 in peritoneum, 9 in blood; CLP at 6, 24, and 72 h (peritoneum and blood): 7, 9, and 7; Sham at 6, 24, and 72 h (peritoneum and blood): 6, 6, and 4. d CD5L − mice were subjected to mid-grade CLP and 3 h later injected IV with 2.5 mg/kg rCD5L, or PBS. Fluids from peritoneum and serum were collected 1 h later and total CD5L was quantified by ELISA. DL, detection limit: 6.25 pg/ml. Mice per group: 4 for PBS; 5 for rCD5L. Pooled data from at least 2 independent experiments ( c , d ). e Scatterplot illustrates the correlation between peritoneal and blood rCD5L levels following IV injection, accompanied by Spearman’s rank correlation coefficient and the two-tailed P value ( n = 5). Statistical differences between groups analyzed by one-way ANOVA with Dunnett’s multiple comparisons correction ( b ), two-tailed unpaired t-tests with Welch’s correction ( c ), or two-tailed Mann-Whitney test ( d ). Floating bars show the minimum, average, and maximum values within each group.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Injection, IV Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Determination of IgM-bound and free endogenous CD5L in peritoneal fluid and serum of WT mice at specified time points after CLP. Values were obtained through densitometric analysis of western blots normalized to baseline IgM-bound CD5L levels (100) in naïve mice. Each group comprised six mice. b ELISA quantification of total IgM in the peritoneum and serum of WT mice at specified times after CLP. Mice per group at 0, 6, 24, and 72 h were as follows: Peritoneum: 7, 9, 7, and 7; Blood: 8, 9, 6, and 7, respectively. c ELISA quantification of total CD5L in the peritoneal cavity of mice subjected to high-grade CLP, and IP- or IV-treated with rCD5L. At 6 h, mice had received one dose of rCD5L (2.5 mg/kg), or PBS, 3 h after CLP. At 24 h, mice had received 2 doses of rCD5L, or PBS, 3 and 6 h after CLP. Mice per group: IP treatment: 6 untreated and 6 treated, at 6 h; 7 untreated and 5 treated, at 24 h. IV treatment: 8 untreated and 6 treated, at 6 h; 8 untreated and 6 treated, at 24 h. d Determination of IgM-bound and free total (endogenous + recombinant) CD5L in the peritoneal cavity of WT mice treated with 2.5 mg/kg rCD5L, or PBS (untreated), via IP or IV routes, 3 h after high-grade CLP, and euthanized 3 h later. Values were obtained by densitometric analysis of western blot bands corresponding to IgM-bound and free CD5L. Mice per group: IP treatment: 6 untreated and 5 treated. IV treatment: 5 untreated and 6 treated. Pooled data from at least 2 independent experiments ( a – d ). Statistical differences between groups analyzed by two-tailed unpaired t-tests with Welch’s correction ( a ), one-way ANOVA with Dunnett’s multiple comparisons correction ( b ), or two-tailed Mann-Whitney test ( c , d ). Floating bars show the minimum, average, and maximum values within each group.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a ELISA quantification of LPS in sera from WT and CD5L − mice 72 h after mid-grade CLP; and in sera from WT mice subjected to high-grade CLP followed by rCD5L treatment. In these, mice were IP- or IV-injected with PBS (untreated), or with two doses of 2.5 mg/kg rCD5L, 3 and 6 h after surgery. Sera was collected and analyzed at 24 h. DL, detection limit: 0.16 ng/ml. The experimental groups comprised 7 WT and 6 CD5L − mice for mid-grade CLP, and for high-grade CLP: 7 IP-untreated, 5 IP-treated, 8 IV-untreated, and 6 IV-treated mice. b , c HMGB1 protein expression in WT and CD5L − organs, determined by immunofluorescence. b Representative images of HMGB1 expression in paraffin-preserved sections of lung, liver, and kidney of WT and CD5L − mice. c Quantification of HMGB1-stained areas in 3 regions from 2 independent images, from 2 mice per CLP group (72 h), or from 1 naïve control mouse per group (0 h), normalized by the DAPI-stained area in the corresponding regions. d , e HMGB1 expression in organs of WT mice subjected to high-grade CLP, and IP- or IV-treated with two doses of rCD5L. d Representative images of HMGB1 expression in lung, liver, and kidney of treated and untreated mice. e Quantification of HMGB1-stained areas, as in ( c ). a , c , e Floating bars represent the minimum, average and maximum values within each group. Statistical differences between groups were analyzed using a two-tailed Mann-Whitney test. b , d Scale bar: 20 μm. f WT and CD5L − mice were IP-injected with a sublethal LPS dose (1.5 mg/kg). Survival was monitored for 6 days. g WT mice were injected with a lethal LPS dose (10 mg/kg), and 3 h later with 2.5 or 5 mg/kg rCD5L, or left untreated (0). Survival was monitored for 4 days. f , g Kaplan–Meier curves were generated to compare survival between groups. Significance was determined by log-rank (Mantel-Cox) test. The graphical representation includes pooled data from 3 independent experiments for ( f ) and 2 independent experiments for g .

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Expressing, Immunofluorescence, Staining, Two Tailed Test, MANN-WHITNEY, Generated

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: a Quantification of inflammatory chemokines in WT and CD5L − mice at indicated time points after mid-grade CLP, using bead-based multiplex immunoassays. b WT mice subjected to high-grade CLP were IV-injected with 2.5 mg/kg of rCD5L, or PBS (untreated), 3 h later. Inflammatory chemokines were quantified 6 h post-CLP. c CXCL1 concentrations in WT and CD5L − mice after mid-grade CLP (extracted from data presented in a ). d CXCL1 concentrations in untreated and IV-treated WT mice after high-grade CLP (extracted from data presented in b ). a – d Pooled data from at least 2 independent experiments, analyzed by two-tailed Mann-Whitney test. a , c Mice per group: Peritoneum: 4 WT and 4 CD5L − at 0 h, 4 WT, and 3 CD5L − at 3 h, 6 WT, and 6 CD5L − at 6 h; Blood, 9 WT and 9 CD5L − at 0 h, 3 WT, and 3 CD5L − at 3 h, 8 WT and 5 CD5L − at 6 h. b , d Mice per group: 6 in untreated, 5 in rCD5L IV-treated. e CD5L − mice were injected IV with rCD5L (2.5 mg/kg) 3 h after mid-grade CLP and peritoneal cells were recovered 3 h later. Percentage of CD5L-bound cells within CD45 - cells; neutrophils (CD45 + CD11b + Ly6G + ), macrophages (CD45 + CD11b + F4/80 + ), other CD11b + (CD45 + CD11b + F4/80 - Ly6G - ), B cells (CD45 + B220 + ), and T cells (CD45 + CD3 + ). f MFI values of the CXCL1 channel within CXCL1 + cells in each subset defined in e . e , f Pooled data from 2 independent experiments, analyzed by two-way ANOVA. Mice per group: 5 untreated and 6 rCD5L-treated. Ly6G and CD11b MFI in neutrophils collected from the peritoneal cavity of rCD5L IP- (left panel) or IV-treated (right panel) WT mice, after high-grade CLP ( g ), or WT vs. CD5L − mice after mid-grade CLP ( h ). Mice per group: IP treatment: 3 at 6 h, 4 at 24 h; IV treatment: 6 at 6 h, 6 at 24 h ( g ); IP and IV treatment: 4 ( h ). Pooled data from 2 independent experiments, statistical comparisons between groups were established by two-tailed unpaired t-tests with Welch’s correction.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Multiplex Assay, Injection, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: CD5L as a promising biological therapeutic for treating sepsis

doi: 10.1038/s41467-024-48360-8

Figure Lengend Snippet: Intravenous administration of rCD5L leads to elevated levels of free bioactive protein in the peritoneal cavity (1). Upon binding to target cells of non-hematopoietic origin (2), rCD5L induces significant production of the CXCL1 chemokine (3). The production of CXCL1 establishes a chemotactic gradient, resulting in heightened neutrophil activation (4) and recruitment from the bloodstream (5). The exact mechanism behind neutrophil activation, whether through increased CXCL1 levels or direct action of rCD5L, remains unclear. Increased neutrophil presence in the peritoneal cavity, coupled with the synergistic effect of rCD5L in bacterial binding and enhanced phagocytosis, facilitates efficient bacterial clearance (6). rCD5L aids in the effective removal of DAMPs, likely from both the bloodstream and the peritoneum (7). Endogenous local release of CD5L from resident macrophages contributes to the overall pool of free CD5L at the site of infection. This illustration is for explanatory purposes and is not drawn to scale. Created with Biorender.com.

Article Snippet: Mouse CD5L quantification was performed using Mouse CD5L ELISA Pair Set (SinoBiological), mouse CXCL1 was quantified using CXCL1/KC DuoSet ELISA (R&D Systems), LPS using Mouse LPS ELISA Kit (Biorbyt Ltd.), AST using mouse AST ELISA Kit (Elabscience), and creatinine using Cr ELISA kit (MyBioSource.com).

Techniques: Binding Assay, Activation Assay, Infection

Journal: Veterinary pathology

Article Title: Flow cytometric analysis of equine bronchoalveolar lavage fluid cells in horses with and without severe equine asthma.

doi: 10.1177/03009858211042588

Figure Lengend Snippet: Figure 5. Flow cytometric analysis of equine bronchoalveolar lavage fluid (BALF) leukocytes. (a) Indicates that cells from p1 have higher forward scatter (FSC) and side scatter (SSC) than cells from p2, indicating they are larger and more internally complex. (b) Unstained control sample showing that many (red circle) but not all cells from p1 (upper panel) have autofluorescence in the phycoerythrin (PE) and fluorescein isothiocyanate (FITC) channels. However, cells from p2 (lower panel) are not autofluorescent. (c) Using anti-CD90 (neutrophil marker) and anti-CD206 (macrophage marker) antibodies, cells from p1 (upper panel) but not p2 (lower panel) are identified as neutrophils (Q3) and macrophages (Q1), respectively. (d) Using antibodies against CD5 (lymphocyte marker) and PanB cells, cells in p1 (upper panel) are double- negative, and cells in p2 (lower panel) are identified as lymphocytes; the red circle indicates autofluorescent cells.

Article Snippet: The fluorescent primary antibodies used in this study were phycoerythrin (PE) conjugated mouse antihuman CD206 (clone 3.29B1, Beckman Coulter), fluorescein isothiocyanate (FITC) conjugated mouse anti-horse CD5 (clone CVS5, Bio-Rad), and PE conjugated mouse anti-horse PanB cells (clone CVS36, Bio-Rad).

Techniques: Control, Marker

Journal: The FASEB Journal

Article Title: A self‐organized actomyosin drives multiple intercellular junction disruption and directly promotes neutrophil recruitment in lipopolysaccharide‐induced acute lung injury

doi: 10.1096/fj.201701506rr

Figure Lengend Snippet: Figure 2. Dynamic alterations of lung TJ and AJ, HUVEC permeability, and neutrophils after LPS administration in vivo and in vitro. E-cadherin (A, A9), VE-cadherin (B, B9), occludin (C, C0), CD11b (E, E9), and MPO (F, F9) staining and quantification of relative intensity or positive cells in 1 alveoli at 1, 3, 5, and 7 d after administration of LPS (15 mg/kg) or saline; and CD3, CD5, CD19, and CD45 staining (G) at 3 d after LPS treatment are shown. D) Double-labeling images are shown of occludin (red) with E-cadherin or VE-cadherin (green) and superimposition with DAPI (blue). H) Percentage of CD11b-, MPO-, CD3-, CD5-, CD19-, and CD45-positive cells in 1 alveoli. I) Levels of IL-6, IL-8, and TNF-a and MPO activity in the supernatants of human neutrophils at 1, 3, 5, 7, and 9 h after LPS (10 mg/ml) treatment are shown. J) HUVEC monolayer permeability, as reflected by FITC-albumin fluorescence intensity, was analyzed at 1, 3, 5, 7, and 9 h after treatment of LPS alone or LPS + neutrophil supernatants (LPS + S). Values are expressed as the means 6 SE; n = 3 (I, J); n = 10–13 alveoli from 3 mice (A2C9, E9,F9, H). Scale bars: 5 mm (D); 30 mm (A–C. E–G). N.S., no significant difference. **P , 0.01 compared with the control group, ##P , 0.01 compared with LPS group at 1 h.

Article Snippet: CD3, CD5, and CD45 antibodies were purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Permeability, In Vivo, In Vitro, Staining, Saline, Labeling, Activity Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Reduced inflammatory cell recruitment and tissue damage in spinal cord injury by acellular spinal cord scaffold seeded with mesenchymal stem cells

doi: 10.3892/etm.2016.3941

Figure Lengend Snippet: Quantitative comparison of the number of positive cells per field for (A) macrophages (CD68) and (B) T lymphocytes (CD5) showing significant differences for the BMSCs + ASC group compared with the ASC group (*P<0.05) and control group ( # P<0.05) at 2 weeks after surgery. (C) IgM shows no significant difference (P>0.05) among the groups. ASC, acellular spinal cord; BMSCs, bone marrow stromal cells; IgM, immunoglobulin M.

Article Snippet: Fluorescein isothiocyanate-labeled primary antibodies against immunoglobulin M (FITC anti-rat IgM; dilution, 1:100; cat. no. RUO 400801; BioLegend, Inc., San Diego, CA, USA), macrophages (anti-CD68/FITC; 100 µg; dilution, 1:100; cat. no. BS-0649R; Bioss, Inc., Woburn, MA, USA), and T lymphocytes (anti-CD5/FITC; dilution, 1:100; cat. no. BS-10218R; Bioss, Inc.) were used to evaluate each section.

Techniques: