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: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) CD5L-KO mice were generated by CRISPR-Cas9 engineering. Schematic representation of the genome editing strategy to silence Cd5l expression by inserting three in-frame stop codons and a frame shift. (B) Quantification by ELISA of CD5L in the sera of C57BL/6 WT and CD5L-KO mice. DL: detection limit (6.25 pg/mL). Data from at least 3 independent experiments (Mann-Whitney test). (C) Intracellular CD5L was detected, after permeabilization of the cells with Triton X100, by staining with goat anti-CD5L polyclonal antibody followed by Alexa 488-coupled donkey anti-goat secondary antibody. Peritoneal macrophages from WT or CD5L-KO healthy mice were identified through staining with anti-mouse F4/80 mAb followed by Alexa 594-coupled anti-rat secondary antibody. DAPI was used as a nuclear counterstaining. Scale bar: 10 μm. (D) WT or CD5L-KO mice were subjected to surgery to expose and ligate the cecum at approximately half the distance between the distal pole and the base of the cecum, and through-and-through puncture with a 21G needle, to induce a mid-grade sepsis condition. N = 15 (each group). Kaplan–Meier survival curves were generated to compare mortality between the two groups and significance was determined by log-rank (Mantel-Cox) test. (E) Body weight loss quantification. ***, p < 0.005.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Generated, CRISPR, Expressing, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Staining

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: WT or CD5L-KO mice were subjected to CLP to induce mid-grade sepsis. For that, animals were subjected to surgery, exposure and ligation of the cecum (corresponding to approximately half the distance between the distal pole and the base of the cecum) and through-and-through puncture with a 21G needle. Control mice (sham) were subjected to the same surgical procedure but without cecum ligation and puncture. Mice were euthanized 6 or 24 h post-surgery. (A) CFU counts from the peritoneal cavity, blood, lung, liver and kidney. (B) Absolute counts assessed by flow cytometry of cellular populations in the peritoneal cavity. Subsets analyzed: leukocytes, CD45 + ; neutrophils, CD45 + CD11b + Ly6G + ; macrophages, CD45 + CD11b + CD11c - F4/80 + . Ly6C and CD206 markers were used to distinguish between M1 and M2 within the macrophage population. (C) Ly6G and CD11b mean fluorescence intensity (MFI) in expressing neutrophils obtained by geometric mean statistics. (D) Lung, liver and kidney tissue sections obtained 24 h post CLP were stained with hematoxylin and eosin. Scale: 100 μm (E) Pathology score of lung, liver and kidney 24 h post CLP in a double blinded analysis. 0, no signs of inflammation; 1, minimal inflammatory signs; 2, mild inflammation; 3, moderate to severe inflammation. (F) The indicated cytokines were quantified by a multiplex bead-based immunoassay in samples from the peritoneal cavity (left panel) and blood serum (right panel). Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05; **, p < 0.01; ns, not statistically significant.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Ligation, Flow Cytometry, Fluorescence, Expressing, Staining, Multiplex Assay, Bead-based Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) Quantification, by ELISA, of CD5L in the peritoneal cavity (left panel) and serum (right panel) of C57BL/6 mice at 0, 6 or 24 h after mid-grade sepsis (CLP) or in sham operated (sham) animals. (B) WT and CD5L-KO naïve healthy mice were subjected to mid-grade CLP and 3 h later were IV administered with recombinant CD5L (rCD5L) at 0 (PBS) or 2.5 mg/Kg (CD5L). Mice were euthanized 1 h after injection to recover the fluids. Amount of CD5L in the peritoneal cavity (left panel) and serum (right panel), quantified by ELISA. (C) Scatter graphs with detailed illustration of the relation between peritoneal and blood CD5L with the indication of Spearman’s rank correlation in both WT and CD5L-KO mice upon CD5L IV injection as detailed above. Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05; **, p < 0.01; ns, not statistically significant.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Injection, IV Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) Percentage of thioglycolate-elicited Ly6G + CD11b + neutrophils among all cells collected from the peritoneal cavity of WT and CD5L-KO mice, determined by flow cytometry. (B) pHrodo™ Red E. coli BioParticles™ were incubated with 0 or 10 μg of rCD5L for 1 h on ice in TBS containing Ca 2+ . After washing, samples were resuspended in Laemmli buffer and heated at 95 °C for 10 min. Western blot detection of CD5L bound to the particles, using mouse anti-His mAb followed by a secondary goat anti-mouse HRP antibody. (C-D) Phagocytosis of pHrodo particles. Neutrophils were cultured in the presence of pHrodo particles pre-coated with 0 or 10 μg of rCD5L. (C) Percentage of Ly6G + CD11b + neutrophils that internalized pHrodo particles, quantified by flow cytometry. (D) MFI values (geometric mean) of pHrodo channel within the pHrodo + neutrophil population. (E) Cecal bacteria were incubated with 0, 2 and 10 μg of rCD5L for 1 h on ice in TBS containing Ca 2+ . Detection of bacteria-bound rCD5L as in (B). (F) Neutrophils from WT or CD5L-KO mice were cultured in the presence of cecal bacteria pre-coated with 0, 2 or 10 μg of rCD5L. After 3 h, cells were washed, lysed and plated for CFU enumeration. (G) Intracellular staining of CD5L in WT or CD5L-KO naïve mouse blood neutrophils (Ly6G + CD11b + ) and monocytes (F4/80 + CD11b + ) was performed after fixation and permeabilization, with goat anti-mCD5L polyclonal antibody followed by Alexa 488-coupled donkey anti-goat secondary antibody and analyzed by flow cytometry. Data from at least 2 independent experiments. Mann-Whitney test did not reveal any statistical difference between relevant groups.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Flow Cytometry, Incubation, Western Blot, Cell Culture, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) CD5L-KO mice were IV injected with 2.5 mg/Kg of rCD5L, and the circulating amount of rCD5L was quantified by ELISA at the indicated time-points. (B) Protocol for analysis of the immune response after CLP and rCD5L IV treatment. Top, analysis at 6 h, mice had been injected IV with 2.5 mg/Kg rCD5L at 3 h post-surgery, or PBS (untreated), and euthanized 3 h later. Bottom, analysis at 24 h, mice were IV injected with 2 doses of 2.5 mg/Kg rCD5L at 3 and 6 h after surgery, or PBS (untreated), followed by euthanasia at 24 h. (C) Absolute counts and frequencies of cellular populations in the peritoneal cavity, measured by flow cytometry. Subsets analyzed: leukocytes, CD45 + ; neutrophils, CD45 + CD11b + Ly6G + ; macrophages, CD45 + CD11b + CD11c - F4/80 + . Ly6C and CD206 markers were used to distinguish between M1 and M2 within the macrophage population. (D) Ly6G and CD11b MFI in expressing neutrophils, obtained by geometric mean statistics. (E) CFU counts of bacteria obtained from the peritoneal cavity and blood, grown in aerobic or anaerobic conditions, or from lung, liver and kidney, grown in aerobiosis. (F) The indicated cytokines were quantified by a multiplex bead-based immunoassay in samples from the peritoneal cavity (upper panels) and blood serum (lower panels). Data from at least 2 independent experiments (Mann-Whitney test). (G) C57BL/6 mice were subjected to CLP to induce highgrade sepsis. At 3 and 6 h after the surgery, mice were injected IV with doses of 2.5 mg/Kg of rCD5L. Kaplan–Meier survival curves were generated to compare mortality between the two groups and significance was determined by log-rank (Mantel-Cox) test. The number of mice per group is indicated (n). *, p < 0.05; ****, p < 0.0001.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Multiplex Assay, Bead-based Assay, MANN-WHITNEY, Generated

Journal: bioRxiv

Article Title: CD5L constraints acute and systemic inflammation and can be a novel potent therapeutic agent against sepsis

doi: 10.1101/2022.03.08.483540

Figure Lengend Snippet: (A) WT and CD5L-KO mice were subjected to CLP to induce mid-grade sepsis, and CXCL1 was quantified in the peritoneal cavity or blood serum 3 h or 6 h after surgery. (B) WT C57BL/6 mice were subjected to CLP to induce high grade sepsis and IV injected with 2.5 mg/Kg of rCD5L, or with PBS (untreated) 3 h after surgery. CXCL1 was quantified by a multiplex bead-based immunoassay in the peritoneal cavity and blood serum 6 h after CLP. Data from at least 2 independent experiments (Mann-Whitney test). *, p < 0.05.

Article Snippet: mCD5L quantification was performed using the Mouse CD5L ELISA Pair Set (SinoBiological) following the manufacturer’s instructions.

Techniques: Injection, Multiplex Assay, Bead-based Assay, MANN-WHITNEY

Journal: Nature Communications

Article Title: Macrophage lineage cells-derived migrasomes activate complement-dependent blood-brain barrier damage in cerebral amyloid angiopathy mouse model

doi: 10.1038/s41467-023-39693-x

Figure Lengend Snippet: a Plasma CD5L concentration of CAA patients ( N = 12) and HC ( N = 12) as well as CAA models ( N = 5 in each group) and WT C57/BL6 mice ( N = 5). Data are presented as mean values ± SEM with the indicated significance (by two-tailed Student’s t test). b Left: CD5L deposition in mice brain was evaluated with immunostaining. Right: Immunostaining of CD31 (red), CD5L (green), C5b-9 (blue) and TSPAN4 (purple). Data are representative of 4 biologically independent experiments. c Plasma C5-9 concentration of mice. N = 6 in PBS-M, Aβ40-M and CD5L KO Aβ40-M transferred mice and N = 5 in CD5L KO PBS-M treated group. Data are presented as mean values ± SEM with the indicated significance (by one-way ANOVA followed by Tukey’s post-test). d C5b-9 deposition in brain as assessed with immunostaining. Data are representative of 3 biologically independent experiments. e Fluorescent intensity of 3kDa-Dextran in the plasma and eyeballs of the recipients. N = 6 in PBS-M, Aβ40-M and CD5L KO Aβ40-M transferred mice and N = 5 in CD5L KO PBS-M treated group. Data are presented as mean values ± SEM with the indicated significance (by one-way ANOVA followed by Tukey’s post-test). f Leakage of 3kDa-Dextran in brain parenchyma was assessed with fluorescent microscopy. Data are representative of 3 biologically independent experiments. g Coronal brain sections of the recipients were subjected to immunostaining of CD31 (green) and ZO-1 (red). Data are representative of 3 biologically independent experiments. h Skin autopsy sample of a CAA patient (Left) and brain tissue of WT and Tg-SwDI/B +/+ mice (24w of age) (Right) were subjected to IEM with CD5L staining. Blue dash lines outline micro-vessel wall. Red stars emphasize blood vessel lumen. Red arrow heads emphasize peri-vessel macrophages. Red arrows emphasize CD5L deposition in blood vessels within migrasome-like structures. Data are representative of 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: Concentration of mouse NEFL (FineTest, EM1688), mouse C5b-9 (FineTest, EM1392), mouse CD5L (Boster, EK1414), human CD5L (MEIMIAN, MM-50587H1), human Aβ40 (CUSABIO, CSB-E08299h), human Aβ42 (MAI Bio, LM-2685H), and human C5b-9 (FineTest, EH3858) in plasma was assessed with commercial kits according to instructions of manufacturers.

Techniques: Clinical Proteomics, Concentration Assay, Two Tailed Test, Immunostaining, Microscopy, Staining

Journal: Nature Communications

Article Title: Macrophage lineage cells-derived migrasomes activate complement-dependent blood-brain barrier damage in cerebral amyloid angiopathy mouse model

doi: 10.1038/s41467-023-39693-x

Figure Lengend Snippet: a Plasma CD5L concentration of CAA patients ( N = 12) and HC ( N = 12) as well as CAA models ( N = 5 in each group) and WT C57/BL6 mice ( N = 5). Data are presented as mean values ± SEM with the indicated significance (by two-tailed Student’s t test). b Left: CD5L deposition in mice brain was evaluated with immunostaining. Right: Immunostaining of CD31 (red), CD5L (green), C5b-9 (blue) and TSPAN4 (purple). Data are representative of 4 biologically independent experiments. c Plasma C5-9 concentration of mice. N = 6 in PBS-M, Aβ40-M and CD5L KO Aβ40-M transferred mice and N = 5 in CD5L KO PBS-M treated group. Data are presented as mean values ± SEM with the indicated significance (by one-way ANOVA followed by Tukey’s post-test). d C5b-9 deposition in brain as assessed with immunostaining. Data are representative of 3 biologically independent experiments. e Fluorescent intensity of 3kDa-Dextran in the plasma and eyeballs of the recipients. N = 6 in PBS-M, Aβ40-M and CD5L KO Aβ40-M transferred mice and N = 5 in CD5L KO PBS-M treated group. Data are presented as mean values ± SEM with the indicated significance (by one-way ANOVA followed by Tukey’s post-test). f Leakage of 3kDa-Dextran in brain parenchyma was assessed with fluorescent microscopy. Data are representative of 3 biologically independent experiments. g Coronal brain sections of the recipients were subjected to immunostaining of CD31 (green) and ZO-1 (red). Data are representative of 3 biologically independent experiments. h Skin autopsy sample of a CAA patient (Left) and brain tissue of WT and Tg-SwDI/B +/+ mice (24w of age) (Right) were subjected to IEM with CD5L staining. Blue dash lines outline micro-vessel wall. Red stars emphasize blood vessel lumen. Red arrow heads emphasize peri-vessel macrophages. Red arrows emphasize CD5L deposition in blood vessels within migrasome-like structures. Data are representative of 3 biologically independent experiments. Source data are provided as a Source Data file.

Article Snippet: Concentration of mouse NEFL (FineTest, EM1688), mouse C5b-9 (FineTest, EM1392), mouse CD5L (Boster, EK1414), human CD5L (MEIMIAN, MM-50587H1), human Aβ40 (CUSABIO, CSB-E08299h), human Aβ42 (MAI Bio, LM-2685H), and human C5b-9 (FineTest, EH3858) in plasma was assessed with commercial kits according to instructions of manufacturers.

Techniques: Clinical Proteomics, Concentration Assay, Two Tailed Test, Immunostaining, Microscopy, Staining

Journal: Nature aging

Article Title: Limited Proteolysis-Mass Spectrometry Reveals Aging-Associated Changes in Cerebrospinal Fluid Protein Abundances and Structures

doi: 10.1038/s43587-022-00196-x

Figure Lengend Snippet: a. Nonreducing Western blot against human CD5L in older adults with no cognitive impairment (NCI) and age-matched patients with AD. Scatter plot shows Western blot intensity versus age in the NCI cohort. Error band: 95% CI for line slope. P value: linear regression.

Article Snippet: Structures of free Cd5l and covalent Cd5l-IgM, known to coexist in plasma. c. Nonreducing Western blot (anti-mCd5l, R&D Systems AF2834) of N=4 young vs. N=4 old biologically distinct CSF pools. rCd5l = recombinant mouse Cd5l (R&D Systems 2834-CL).

Techniques: Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Apoptosis Inhibitor of Macrophages Contributes to the Chronicity of Mycobacterium avium Infection by Promoting Foamy Macrophage Formation.

doi: 10.4049/jimmunol.2200306

Figure Lengend Snippet: FIGURE 6. Histopathological characteristics and accumulation of FMs in recombinant AIM-treated mice after M. avium infection. (A) Based on the total lung cell count, the absolute numbers of AMs and IMs were calculated according to Fig. 2B. Bars, mean ± SD (n 5 34/group). Results were confirmed using three independent experiments. (B) MFI of Pacific Blue in FMs from mice at 4 wk after infection. Bars, mean ± SD (n 5 34/group). Results were confirmed using three independent experiments. (C) Representative images of H&E staining of mouse lungs 4 wk after infection. Upper image, low-power field (40×); lower image, high-power field (400×). Scale bars, 200 and 20 mm in low- and high-power fields, respectively. (D) Representative images of Oil Red O staining of mouse lungs 4 wk after infection. Upper image, low-power field (100×); lower image, high-power field (400×). Scale bars, 100 and 20 mm in low- and high-power fields, respectively. Results were con- firmed using two independent experiments. (E) For quantification of Oil Red Opositive cells in the lungs, positive areas were evaluated with ImageJ Fiji software at ×400 magnification. Assessments were made in at least six randomly selected separate regions for each mouse. Bars, mean ± SD (n 5 3/group). (F) mRNA levels of Nceh1, Abca1, Abcg1, and Acat1 in the lungs at 4 wk after infection. Data are expressed as fold increase. Bars, mean ± SD (n 5 34/group). Results were confirmed using three independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: In some experiments, 1 mg recombinant AIM (2834-CL; R&D Systems, Minneapolis, MN) was administered intranasally or i.v. to mice once per week starting on the day of infection.

Techniques: Recombinant, Infection, Cell Counting, Staining, Software

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19 associates with CD5L to form a putative heterodimer of p19/CD5L. ( a ) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC and cultured for 3 days, and total cell lysates or culture supernatants were immunoprecipitated with anti-FLAG or anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-c-MYC or biotin-conjugated anti-p19, respectively. Immunoprecipitated protein was confirmed by western blotting with the antibody used for immunoprecipitation. ( b ) HEK293T cells were transiently transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, or both and p3 × FLAG-CMV-14-Hyper-p19/CD5L, then cultured for 3 days. The culture supernatants were then subjected to p19/CD5L-specific ELISA in triplicate. Data are shown as the mean ± SD. ( c ) Naive CD4 + T cells were stimulated with plate-coated anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) under pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 8 times, and subjected to immunoprecipitation with anti-CD5L followed by western blotting with biotin-conjugated anti-p19 and subsequently anti-CD5L. ( d ) Naive CD4 + T cells were also similarly stimulated with plate-coated anti-CD3 and anti-CD28 under Th, Th0 and pathogenic Th17-polarizing conditions for 3 days. Resultant culture supernatants were collected and concentrated by centrifugation using a centrifugal filter approximately 10 times, followed by p19/CD5L-specific ELISA in triplicate. ( e ) HEK293-F cells were transiently cotransfected with p3 × FLAG-CMV-14-p19 WT or p3 × FLAG-CMV-14-p19 C55S and pCMV3-CD5L-c-MYC and cultured for 3 days. The culture supernatants were immunoprecipitated with anti-c-MYC and control antibody followed by western blotting with biotin-conjugated anti-p19, then anti-c-MYC. Data are shown as the mean ± SD and representative of four ( a ) or two ( b – e ) independent experiments. P values were determined using one-way ANOVA. * P < 0.05.

Article Snippet: Mouse p19, p40, and EBI3 cDNAs were isolated by RT-PCR using total RNA prepared from concanavalin A-activated spleen cells, confirmed by sequencing, and subcloned into p3 × FLAG-CMV-14 vectors (Sigma-Aldrich), which has 3 × FLAG-epitope-tag sequence at C-terminal, or pCXN2 . pCMV3-CD5L-c-MYC was purchased from Sino Biological.

Techniques: Cell Culture, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Centrifugation

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: CD5L-deficient mice show alleviated EAE with reduced frequency of GM-CSF + CD4 + T cells in the CNS. ( a – d ) WT mice or CD5L-deficient mice were immunized with MOG 35-55 peptide and their clinical scores were monitored with time ( a ). On day 15, spinal cords and brains were harvested, and the CNS was histopathologically analyzed with H&E staining. Representative images are shown ( b ). Mononuclear cells were also isolated from the CNS, and intracellular cytokine staining was performed after restimulation with PMA and ionomycin. Representative dot plots of GM-CSF, and IL-17A in CD4 + T cells are shown ( c ). Average frequencies of respective CD4 + T cells were calculated and compared ( d ). Blood was also taken over time and serum levels of p19/CD5L and CD5L were determined by ELISA ( e , f ). Data are shown as mean ± SD (n = 4–6) and are representative of three independent experiments. P values were determined using unpaired, two-tailed Student’s t -test ( a , d ) or one-way ANOVA ( e , f ). * P < 0.05, ** P < 0.01, *** P < 0.001. NS, not significant.

Article Snippet: Mouse p19, p40, and EBI3 cDNAs were isolated by RT-PCR using total RNA prepared from concanavalin A-activated spleen cells, confirmed by sequencing, and subcloned into p3 × FLAG-CMV-14 vectors (Sigma-Aldrich), which has 3 × FLAG-epitope-tag sequence at C-terminal, or pCXN2 . pCMV3-CD5L-c-MYC was purchased from Sino Biological.

Techniques: Staining, Isolation, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: Differentiation into GM-CSF-producing CD4 + T cells is impaired in CD5L-deficient CD4 + T cells in vitro. Naive CD4 + T cells from WT mice or CD5L-deficient mice were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) for 4 days under various Th-polarizing conditions; Th, Th0, Th1, ThGM, non-pathogenic Th17, and pathogenic Th17. These cells were then restimulated with PMA and ionomycin, and the intracellular cytokine staining was performed. Representative dot plots for GM-CSF, IL-17A, IFN-γ, and IL-10 in CD4 + T cells are shown ( a ), and average frequencies of respective CD4 + T cells were calculated and compared ( b ). Data are shown as mean ± SD (n = 3) and are representative of three independent experiments. P values were determined using unpaired, two-tailed Student’s t -test. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mouse p19, p40, and EBI3 cDNAs were isolated by RT-PCR using total RNA prepared from concanavalin A-activated spleen cells, confirmed by sequencing, and subcloned into p3 × FLAG-CMV-14 vectors (Sigma-Aldrich), which has 3 × FLAG-epitope-tag sequence at C-terminal, or pCXN2 . pCMV3-CD5L-c-MYC was purchased from Sino Biological.

Techniques: In Vitro, Staining, Two Tailed Test

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19/CD5L but not either alone efficiently induces cell proliferation, STAT5 phosphorylation. ( a , b ) HEK293T cells were transfected with the control vector p3 × FLAG-CMV-14 alone, p3 × FLAG-CMV-14-p19, pCMV3-CD5L-c-MYC, p3 × FLAG-CMV-14-hyper-p19/CD5L, and p3 × FLAG-CMV-13-hyper-p40/CD5L. The cells were also cotransfected with expression vectors of p3 × FLAG-CMV-14-p19 and pCMV3-CD5L-c-MYC, together with those of p3 × FLAG-CMV-14-p19 and p3 × FLAG-CMV-14-p40, which produced IL-23 as the positive control. The total amount of DNA in each transfection sample was adjusted to be kept equal with the empty vector. Three days later, culture supernatants were collected, and 10% or 30% of them were used for stimulation of Ba/F3 cells expressing gp130/IL-12Rβ1/IL-12Rβ2/IL-23Rα. For p19 + p40 (IL-23), only 10% culture supernatant was added; therefore, “ – ” means that no data exist for the 30% lane ( a ). Ba/F3 cells were also stimulated with purified recombinant p19, CD5L, a mixture of p19 and CD5L (all 20 ng/ml), and hyper-p19/CD5L (0.1 – 20 ng/ml) ( b ). Proliferative activity of these cells was determined by measuring 3 H-thymidine incorporated into the DNA. ( c , d ) Naive CD4 + T cells from WT mice were stimulated with plate-coated anti-CD3 (2 μg/ml ) and anti-CD28 (1 μg/ml ) for 3 days under Th conditions, washed, and rested in 10% FBS medium for 6 h. These cells were unrestimulated ( – ) or then restimulated with purified recombinant p19, CD5L, a mixture of p19 and CD5L, hyper-p19/CD5L, IL-27 (positive control for phosphorylation of STAT1 and STAT3, all 20 ng/ml), or IL-2 (positive control for phosphorylation of STAT5, 100 U/ml) for 5, 15 and 60 min, and subjected to western blotting with anti-pY-STATs and subsequently anti-total STATs ( c ). FACS analysis was also performed using anti-pY-STAT5 and its control antibody after stimulation for 20 min ( d ). Data are shown as the mean ± SD in triplicate and are representative of three ( a , c , d ) or two ( b ) independent experiments. P values were determined using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mouse p19, p40, and EBI3 cDNAs were isolated by RT-PCR using total RNA prepared from concanavalin A-activated spleen cells, confirmed by sequencing, and subcloned into p3 × FLAG-CMV-14 vectors (Sigma-Aldrich), which has 3 × FLAG-epitope-tag sequence at C-terminal, or pCXN2 . pCMV3-CD5L-c-MYC was purchased from Sino Biological.

Techniques: Transfection, Plasmid Preparation, Expressing, Produced, Positive Control, Purification, Recombinant, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: IL-23p19 and CD5 antigen-like form a possible novel heterodimeric cytokine and contribute to experimental autoimmune encephalomyelitis development

doi: 10.1038/s41598-021-84624-9

Figure Lengend Snippet: p19/CD5L but not either alone induces augmentation of GM-CSF expression. Naive CD4 + T cells from WT mice were stimulated with plate-coated anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) in the presence of recombinant p19, CD5L (all 20 ng/ml), and hyper-p19/CD5L (2–20 ng/ml) under Th conditions for 3 days, and subjected to intracellular staining of GM-CSF and IFN-γ ( a , b ). Representative dot plots were shown ( a ). Culture supernatants were analyzed for GM-CSF by ELISA ( c ). Similarly, naive CD4 + T cells from CD5L-deficient mice were stimulated and subjected to intracellular staining of GM-CSF and IFN-γ ( d , e ). Representative dot plots were shown ( d ). Culture supernatants were analyzed for GM-CSF by ELISA ( f ). Data are shown as mean ± SD in triplicates and are representative of more than two independent experiments. P values were determined using one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Mouse p19, p40, and EBI3 cDNAs were isolated by RT-PCR using total RNA prepared from concanavalin A-activated spleen cells, confirmed by sequencing, and subcloned into p3 × FLAG-CMV-14 vectors (Sigma-Aldrich), which has 3 × FLAG-epitope-tag sequence at C-terminal, or pCXN2 . pCMV3-CD5L-c-MYC was purchased from Sino Biological.

Techniques: Expressing, Recombinant, Staining, Enzyme-linked Immunosorbent Assay