Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: (A) SPR between rmCIRP and rmTREM-1. Anti-his Ab was used to capture rmCIRP-his. rmTREM-1 was injected as an analyte in concentrations of 0 to 500 nM. (B) 1.5 × 10 4 RAW264.7 cells were treated with rmCIRP (5 μg/ml) at 4°C for 10 min, fixed in a nonpermeabilized fashion, and stained with rabbit anti-mouse CIRP Ab, goat anti-mouse TREM-1 Ab, goat anti-CD11b Ab, Cy5-conjugated AffiniPure donkey anti-goat IgG, and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG. Confocal microscopy images were obtained at using a Zeiss LSM880 confocal microscope equipped with a 63× objective. Images were analyzed and quantified by using the ZenBlue software. ( C) After the staining protocol described in (B) , cell associated fluorescence was measured on Biotek Synergy Neo2 at 566 nm upon excitation at 488 nm ( E 1), at 681 nm after excitation at 630 nm ( E 2), and at 681 nm after excitation at 488 nm ( E 3). The transfer of fluorescence was calculated as FRET units. FRET unit = [ E 3 both − E 3 none ] − [( E 3 Cy5 − E 3 none ) × ( E 2 both / E 2 Cy5 )] − [( E 3 Cy3 − E 3 none ) × ( E 1 both / E 1 Cy3 )]. Data are expressed as means ± SE obtained from three independent experiments, n=9/group. Groups compared by unpaired t-test (*p<0.01 vs. CD11b). (D) A total of 1 × 10 6 /ml RAW264.7 cells were stimulated with rmCIRP (1 µg/ml) for various times. Extracted proteins were immunoprecipitated by using anti-DAP12 Ab, followed by Western blotting using pTyr (4G10) and DAP12 Ab. Extracted proteins obtained from rmCIRP (1 µg/ml for various times) stimulated RAW264.7 cells (1 × 10 6 /ml) were subjected to Western blotting using pSyk, Syk, and β–actin Abs. Representative western blots for phosphotyrosine (4G10), DAP12, pSyk, Syk, and β–actin are shown. (E, F) Each blot was quantified by densitometric analysis. Phosphotyrosine (pDAP12) and pSyk expression in each sample was normalized to DAP12 or Syk or β-actin expression and the mean values of 0 min of rmCIRP-treated groups were standardized as one for comparison. Data are expressed as means ± SE (n=6 samples/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. rmCIRP at 0 min). (G) RAW264.7 cells were transfected with TREM-1 siRNA, control siRNA, underwent mock transfection, or no transfection. Cells were then stimulated with PBS control or 1μg/ml rmCIRP. After 6 h, TNF-α in the supernatant was analyzed by ELISA. Data are expressed as means ± SE (n=3 samples/group). Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs. respective PBS group; # p<0.01 vs. rmCIRP-treated non-transfected cells). (H) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and stimulated with PBS or rmCIRP (1 µg/ml). Simultaneously cells were treated with various doses of LP-17 or LP-17-Sc1. After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from five independent experiments (n=3-10 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS; #p<0.05 vs. rmCIRP+PBS). FRET, fluorescence resonance energy transfer; TNF, tumor necrosis factor; DAP12, DNAX activation protein of 12kDa; ELISA, enzyme-linked immunosorbent assay; PBS phosphate buffered saline.

Article Snippet: Antibodies used were as follows: rabbit anti-mouse CIRP Ab (Catalog 10209-2-AP; ProteinTech, Rosemont, IL), goat anti-mouse TREM-1 Ab (Catalog AF1187; R&D Systems), goat anti-CD11b Ab (Catalog MBS420973MyBioSource, San Diego, CA), Cy5-conjugated AffiniPure donkey anti-goat IgG (Code 705-175-147; Jackson ImmunoResearch Laboratories, West Grove, PA) and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (Code 711-166-152; Jackson ImmunoResearch Laboratories).

Techniques: Injection, Staining, Confocal Microscopy, Microscopy, Software, Fluorescence, Immunoprecipitation, Western Blot, Expressing, Comparison, Transfection, Enzyme-linked Immunosorbent Assay, Förster Resonance Energy Transfer, Activation Assay, Saline

Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: (A) Partial amino acid sequence of murine CIRP highlighting an area of similarity between murine PGLYRP1. Three peptides (M1, M2, and M3) are highlighted from within the CIRP sequence. RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with 10 µg/ml of peptides M1, M2, or M3 for 30 min. Cells were then stimulated with rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from two independent experiments (n=6 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. unstimulated cells and #p<0.05 vs. rmCIRP-treated cells). (B) SPR between rmTREM-1 and M3. M3 was injected as an analyte in concentrations of 0 to 20 μM. (C) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with M3 or M3-Sc1 at a dose of 10 µg/ml for 30 min. Cells were then stimulated with PBS or rmCIRP (5 µg/ml) at 4°C for 10 min, fixed in a nonpermeabilized fashion, and stained as described in 1B. FRET analysis was performed as described in 1B. Data are expressed as means ± SE obtained from three independent experiments, n=9/group. Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs CD11b + rmCIRP, #p<0.05 vs TREM-1 + rmCIRP). (D) To activate RAW264.7 cells through TREM-1, 96-well flat bottom plates were pre-coated with 20 μg/ml of an agonist anti-TREM-1 mAb overnight at 37°C. The wells were washed with sterile PBS and 5 × 10 4 cells/well were plated. Prior to plating, cells were premixed with either PBS control, M3 (10 μg/ml) or scramble M3-Sc1 (10 μg/ml) for 30 min. After plating, TNF-α production was measured in the culture supernatants after an additional 24 h of incubation. Data are expressed as means ± SE. The experiment was performed two independent times with n=5 wells per group. Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs. uncoated; #p<0.05 vs. TREM-1 Ab + PBS). (E) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with various doses of M3, M3-Sc1, or M3-Sc1 for 30 min. Cells were then stimulated with PBS or rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from three independent experiments (n=4 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated cells; #p<0.05 vs. rmCIRP + PBS). (F) Macrophages from healthy human donors (1×10 4 cells/ml) were plated in 96-well culture plate and treated with various doses of M3 for 30 minutes. Cells were then stimulated with PBS or rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE (n=5 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated cells; #p<0.05 vs. rmCIRP + PBS). FRET, fluorescence resonance energy transfer; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate buffered saline; PGLYRP1, peptidoglycan recognition protein 1.

Article Snippet: Antibodies used were as follows: rabbit anti-mouse CIRP Ab (Catalog 10209-2-AP; ProteinTech, Rosemont, IL), goat anti-mouse TREM-1 Ab (Catalog AF1187; R&D Systems), goat anti-CD11b Ab (Catalog MBS420973MyBioSource, San Diego, CA), Cy5-conjugated AffiniPure donkey anti-goat IgG (Code 705-175-147; Jackson ImmunoResearch Laboratories, West Grove, PA) and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (Code 711-166-152; Jackson ImmunoResearch Laboratories).

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Injection, Staining, Sterility, Incubation, Fluorescence, Förster Resonance Energy Transfer, Saline

Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: Sepsis and I/R causes an increased release of eCIRP. As the endogenous ligand eCIRP recognizes TREM-1 and activates intracellular signaling molecules DAP12 and Syk, leading to increased expression of pro-inflammatory mediators that cause excessive inflammation and remote tissue injury. eCIRP increases TREM-1 expression, possibly via positive feedback induction. A small peptide M3 derived from human eCIRP abrogates eCIRP-TREM-1 interaction, thereby leading to decreased inflammation and attenuated ALI. I/R, ischemia and reperfusion; DAP12, DNAX activation protein of 12kDa; ALI, acute lung injury.

Article Snippet: Antibodies used were as follows: rabbit anti-mouse CIRP Ab (Catalog 10209-2-AP; ProteinTech, Rosemont, IL), goat anti-mouse TREM-1 Ab (Catalog AF1187; R&D Systems), goat anti-CD11b Ab (Catalog MBS420973MyBioSource, San Diego, CA), Cy5-conjugated AffiniPure donkey anti-goat IgG (Code 705-175-147; Jackson ImmunoResearch Laboratories, West Grove, PA) and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG (Code 711-166-152; Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Derivative Assay, Activation Assay

Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: (A) SPR between rmCIRP and rmTREM-1. Anti-his Ab was used to capture rmCIRP-his. rmTREM-1 was injected as an analyte in concentrations of 0 to 500 nM. (B) 1.5 × 10 4 RAW264.7 cells were treated with rmCIRP (5 μg/ml) at 4°C for 10 min, fixed in a nonpermeabilized fashion, and stained with rabbit anti-mouse CIRP Ab, goat anti-mouse TREM-1 Ab, goat anti-CD11b Ab, Cy5-conjugated AffiniPure donkey anti-goat IgG, and Cy3-conjugated AffiniPure F(ab’)2 fragment donkey anti-rabbit IgG. Confocal microscopy images were obtained at using a Zeiss LSM880 confocal microscope equipped with a 63× objective. Images were analyzed and quantified by using the ZenBlue software. ( C) After the staining protocol described in (B) , cell associated fluorescence was measured on Biotek Synergy Neo2 at 566 nm upon excitation at 488 nm ( E 1), at 681 nm after excitation at 630 nm ( E 2), and at 681 nm after excitation at 488 nm ( E 3). The transfer of fluorescence was calculated as FRET units. FRET unit = [ E 3 both − E 3 none ] − [( E 3 Cy5 − E 3 none ) × ( E 2 both / E 2 Cy5 )] − [( E 3 Cy3 − E 3 none ) × ( E 1 both / E 1 Cy3 )]. Data are expressed as means ± SE obtained from three independent experiments, n=9/group. Groups compared by unpaired t-test (*p<0.01 vs. CD11b). (D) A total of 1 × 10 6 /ml RAW264.7 cells were stimulated with rmCIRP (1 µg/ml) for various times. Extracted proteins were immunoprecipitated by using anti-DAP12 Ab, followed by Western blotting using pTyr (4G10) and DAP12 Ab. Extracted proteins obtained from rmCIRP (1 µg/ml for various times) stimulated RAW264.7 cells (1 × 10 6 /ml) were subjected to Western blotting using pSyk, Syk, and β–actin Abs. Representative western blots for phosphotyrosine (4G10), DAP12, pSyk, Syk, and β–actin are shown. (E, F) Each blot was quantified by densitometric analysis. Phosphotyrosine (pDAP12) and pSyk expression in each sample was normalized to DAP12 or Syk or β-actin expression and the mean values of 0 min of rmCIRP-treated groups were standardized as one for comparison. Data are expressed as means ± SE (n=6 samples/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. rmCIRP at 0 min). (G) RAW264.7 cells were transfected with TREM-1 siRNA, control siRNA, underwent mock transfection, or no transfection. Cells were then stimulated with PBS control or 1μg/ml rmCIRP. After 6 h, TNF-α in the supernatant was analyzed by ELISA. Data are expressed as means ± SE (n=3 samples/group). Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs. respective PBS group; # p<0.01 vs. rmCIRP-treated non-transfected cells). (H) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and stimulated with PBS or rmCIRP (1 µg/ml). Simultaneously cells were treated with various doses of LP-17 or LP-17-Sc1. After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from five independent experiments (n=3-10 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS; #p<0.05 vs. rmCIRP+PBS). FRET, fluorescence resonance energy transfer; TNF, tumor necrosis factor; DAP12, DNAX activation protein of 12kDa; ELISA, enzyme-linked immunosorbent assay; PBS phosphate buffered saline.

Article Snippet: To detect TREM-1 expression on the surface of macrophages, a total of 1 × 10 6 RAW264.7 or primary peritoneal macrophages were washed with FACS buffer containing PBS with 2% FBS and stained with APC anti-mouse TREM-1 Ab (clone: 174021, R&D systems).

Techniques: Injection, Staining, Confocal Microscopy, Microscopy, Software, Fluorescence, Immunoprecipitation, Western Blot, Expressing, Comparison, Transfection, Enzyme-linked Immunosorbent Assay, Förster Resonance Energy Transfer, Activation Assay, Saline

Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: (A) Partial amino acid sequence of murine CIRP highlighting an area of similarity between murine PGLYRP1. Three peptides (M1, M2, and M3) are highlighted from within the CIRP sequence. RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with 10 µg/ml of peptides M1, M2, or M3 for 30 min. Cells were then stimulated with rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from two independent experiments (n=6 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. unstimulated cells and #p<0.05 vs. rmCIRP-treated cells). (B) SPR between rmTREM-1 and M3. M3 was injected as an analyte in concentrations of 0 to 20 μM. (C) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with M3 or M3-Sc1 at a dose of 10 µg/ml for 30 min. Cells were then stimulated with PBS or rmCIRP (5 µg/ml) at 4°C for 10 min, fixed in a nonpermeabilized fashion, and stained as described in 1B. FRET analysis was performed as described in 1B. Data are expressed as means ± SE obtained from three independent experiments, n=9/group. Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs CD11b + rmCIRP, #p<0.05 vs TREM-1 + rmCIRP). (D) To activate RAW264.7 cells through TREM-1, 96-well flat bottom plates were pre-coated with 20 μg/ml of an agonist anti-TREM-1 mAb overnight at 37°C. The wells were washed with sterile PBS and 5 × 10 4 cells/well were plated. Prior to plating, cells were premixed with either PBS control, M3 (10 μg/ml) or scramble M3-Sc1 (10 μg/ml) for 30 min. After plating, TNF-α production was measured in the culture supernatants after an additional 24 h of incubation. Data are expressed as means ± SE. The experiment was performed two independent times with n=5 wells per group. Multiple groups were compared by one-way ANOVA and Tukey method (*p<0.05 vs. uncoated; #p<0.05 vs. TREM-1 Ab + PBS). (E) RAW264.7 cells (1×10 4 cells/ml) were plated in 96-well culture plate and treated with various doses of M3, M3-Sc1, or M3-Sc1 for 30 min. Cells were then stimulated with PBS or rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE obtained from three independent experiments (n=4 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated cells; #p<0.05 vs. rmCIRP + PBS). (F) Macrophages from healthy human donors (1×10 4 cells/ml) were plated in 96-well culture plate and treated with various doses of M3 for 30 minutes. Cells were then stimulated with PBS or rmCIRP (1 µg/ml). After 24 h, TNF-α in culture supernatants at protein level were measured by ELISA. Data are expressed as means ± SE (n=5 wells/group). The groups were compared by one-way ANOVA and SNK method (*p<0.05 vs. PBS-treated cells; #p<0.05 vs. rmCIRP + PBS). FRET, fluorescence resonance energy transfer; TNF, tumor necrosis factor; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate buffered saline; PGLYRP1, peptidoglycan recognition protein 1.

Article Snippet: To detect TREM-1 expression on the surface of macrophages, a total of 1 × 10 6 RAW264.7 or primary peritoneal macrophages were washed with FACS buffer containing PBS with 2% FBS and stained with APC anti-mouse TREM-1 Ab (clone: 174021, R&D systems).

Techniques: Sequencing, Enzyme-linked Immunosorbent Assay, Injection, Staining, Sterility, Incubation, Fluorescence, Förster Resonance Energy Transfer, Saline

Journal: bioRxiv

Article Title: Extracellular CIRP as a Novel Endogenous TREM-1 Ligand to Fuel Inflammation

doi: 10.1101/674218

Figure Lengend Snippet: Sepsis and I/R causes an increased release of eCIRP. As the endogenous ligand eCIRP recognizes TREM-1 and activates intracellular signaling molecules DAP12 and Syk, leading to increased expression of pro-inflammatory mediators that cause excessive inflammation and remote tissue injury. eCIRP increases TREM-1 expression, possibly via positive feedback induction. A small peptide M3 derived from human eCIRP abrogates eCIRP-TREM-1 interaction, thereby leading to decreased inflammation and attenuated ALI. I/R, ischemia and reperfusion; DAP12, DNAX activation protein of 12kDa; ALI, acute lung injury.

Article Snippet: To detect TREM-1 expression on the surface of macrophages, a total of 1 × 10 6 RAW264.7 or primary peritoneal macrophages were washed with FACS buffer containing PBS with 2% FBS and stained with APC anti-mouse TREM-1 Ab (clone: 174021, R&D systems).

Techniques: Expressing, Derivative Assay, Activation Assay