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: Acta neuropathologica

Article Title: TREM2 expression in the brain and biological fluids in prion diseases.

doi: 10.1007/s00401-021-02296-1

Figure Lengend Snippet: Fig. 3 mRNA Trem2 expression in the brain of sCJD MM1 and VV2 mouse models. RT-qPCR analysis Trem2 expression in mouse models of sCJD MM1 (tg340-CJD MM1) (left panels) and sCJD VV2 (tg361-CJD VV2) (right panels) subtypes. Trem2 expression was analysed in five brain regions: anterior and posterior cortex, cer- ebellum, olfactory bulb, and brainstem, at three time points: pre-clinical (80 days post inoculation (dpi)), early-clinical (120 dpi), and clinical (180 dpi). CJD-inoculated animals (black columns) were compared to their corresponding control- inoculated animals (white columns). Four to eight animals per time point and condition were used. Normalization was performed using the house- keeping gene Hprt; *p < 0.05, **p < 0.01

Article Snippet: Human TREM2 goat polyclonal antibody (RD Systems, AF1828) was used diluted 1:20 for immunohistochemistry and at a dilution of 1:200 for western blotting.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Scientific Reports

Article Title: Idelalisib impairs TREM-1 mediated neutrophil inflammatory responses

doi: 10.1038/s41598-018-23808-2

Figure Lengend Snippet: Idelalisib impairs human neutrophil functions in vitro . PMN from healthy human donors were isolated and preincubated with idelalisib (1 µg/ml) or vehicle (DMSO) for 30 min at 37 °C. Cells were activated with indicated stimulus (anti-TREM-1 mAb, isotype matched control mAb, LPS at 1 µg/ml) and neutrophil activation was detected as oxidative burst kinetics ( A,B ) for 3 h. One out of five experiments is depicted. Further samples were activated for 1 h at 37 °C and analyzed by flow cytometry. Degranulation was detected as enhanced surface expression of ( C ) CD11b (n = 5) and ( D ) CD66b (n = 5) surface expression and ( E ) CD62L shedding (n = 4). For IL-8 release cells were incubated with indicated stimuli for 16 h and ( G ) IL-8 was measured out of cell supernatants by standard ELISA (n = 3). (*) indicates a significant difference (p < 0.05) by two-way ANOVA with Bonferroni’s posttest. Mean plus SD is depicted.

Article Snippet: Cross-linking of anti-TREM-1 was performed with a secondary goat anti-mouse F(ab’)2 (Dianova, Hamburg, Germany) for experiments followed by Ca 2+ mobilization assay.

Techniques: In Vitro, Isolation, Activation Assay, Flow Cytometry, Expressing, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Idelalisib impairs TREM-1 mediated neutrophil inflammatory responses

doi: 10.1038/s41598-018-23808-2

Figure Lengend Snippet: Idelalisib impairs calcium flux and TREM-1 signaling in neutrophils in vitro . PMN from healthy human donors were isolated, preincubated with idelalisib 1 µg/ml and loaded with FLUO-3/AM (both at 37 °C for 30 min) for calcium flux assay. ( A – C ) Fluorescence signals were detected by flow cytometry for 30 s. For PMN activation indicated stimuli (( A ) anti-TREM-1, ( B ) isotype matched control mAb, ( C ) ionomycin) was added. For cell stimulation via TREM-1 receptor (and isotype control) cross-linking was performed with a secondary antibody again after 30 s; fluorescence signals were gained for further 5 min. Ionomycin served as positive control. One out of three experiments is depicted. ( D ) For protein analysis PMN were activated with indicated stimuli (anti-TREM-1 antibody, matched control mAb, LPS) for 30 min after preincubated with idelalisib as described above. Proteins were extracted with an urea-based lysis buffer, SDS PAGE was performed and proteins were blotted by a semi-dry process. Protein activation was analyzed by staining of phosphorylated and non-phosphorylated proteins. ß-actin served as loading control. One out of three experiments is depicted.

Article Snippet: Cross-linking of anti-TREM-1 was performed with a secondary goat anti-mouse F(ab’)2 (Dianova, Hamburg, Germany) for experiments followed by Ca 2+ mobilization assay.

Techniques: In Vitro, Isolation, Calcium Flux Assay, Fluorescence, Flow Cytometry, Activation Assay, Cell Stimulation, Positive Control, Lysis, SDS Page, Staining

Journal: Scientific Reports

Article Title: Idelalisib impairs TREM-1 mediated neutrophil inflammatory responses

doi: 10.1038/s41598-018-23808-2

Figure Lengend Snippet: Impaired TREM-1 activation in neutrophils is not specific to PI3Kδ. PMN from healthy human donors were isolated and preincubated with respective PI3K inhibitors 1 µg/ml for 30 min at 37 °C. Cells were activated with anti-TREM-1 antibody and ( A ) oxidative burst was assessed via fluorescence kinetic. Depicted is the SFI after 2 h (n = 5). Further samples were stimulated for 1 h and neutrophil activation was detected as degranulation: ( B ) CD11b (n = 5) and ( C ) CD66b (n = 5) surface expression and ( D ) CD62L shedding (n = 5) by flow cytometry. (*) indicates a significant difference (p < 0.05) by one-way ANOVA with Bonferroni’s posttest. Mean plus SD is depicted.

Article Snippet: Cross-linking of anti-TREM-1 was performed with a secondary goat anti-mouse F(ab’)2 (Dianova, Hamburg, Germany) for experiments followed by Ca 2+ mobilization assay.

Techniques: Activation Assay, Isolation, Fluorescence, Expressing, Flow Cytometry

Journal: Scientific Reports

Article Title: Idelalisib impairs TREM-1 mediated neutrophil inflammatory responses

doi: 10.1038/s41598-018-23808-2

Figure Lengend Snippet: Impaired neutrophil functions in idelalisib-treated patients after ex vivo activation. PMN from idelalisib-treated patients (n = 5) and healthy human donors (n = 5) were isolated. Cells were activated as indicated (anti-TREM-1 antibody, matched control mAb, LPS) for 1 h at 37 °C and neutrophil functions were assessed via flow cytometry. Mean and SD of ( A ) oxidative burst, ( B ) phagocytosis, degranulation – ( C ) CD11b and ( D ) CD66b surface expression and ( E ) CD62L shedding are depicted. (*) indicates a significant difference (p < 0.05) by two-way ANOVA with Bonferroni’s posttest.

Article Snippet: Cross-linking of anti-TREM-1 was performed with a secondary goat anti-mouse F(ab’)2 (Dianova, Hamburg, Germany) for experiments followed by Ca 2+ mobilization assay.

Techniques: Ex Vivo, Activation Assay, Isolation, Flow Cytometry, Expressing

Journal: Scientific Reports

Article Title: Idelalisib impairs TREM-1 mediated neutrophil inflammatory responses

doi: 10.1038/s41598-018-23808-2

Figure Lengend Snippet: Neutrophil functions are restored after discontinuation of idelalisib treatment. PMN from one patient during idelalisib therapy and after discontinuation (d/c) of treatment were isolated. Cells were activated with anti-TREM-1 antibody for 1 h at 37 °C and neutrophil functions were assessed via flow cytometry. Mean and SD of oxidative burst, phagocytosis, degranulation (CD11b and CD66b) and CD62L shedding are depicted.

Article Snippet: Cross-linking of anti-TREM-1 was performed with a secondary goat anti-mouse F(ab’)2 (Dianova, Hamburg, Germany) for experiments followed by Ca 2+ mobilization assay.

Techniques: Isolation, Flow Cytometry

Journal: PLoS ONE

Article Title: TLR-2/TLR-4 TREM-1 Signaling Pathway Is Dispensable in Inflammatory Myeloid Cells during Sterile Kidney Injury

doi: 10.1371/journal.pone.0068640

Figure Lengend Snippet: ( A ) Schema showing experimental design. Mice were subjected to unilateral ischemia and reperfusion injury (U-IRI) and treated daily with 40 µg/mouse of TREM1-Fc or hIgG, as control. ( B ) Western blot showing presence of TREM1-Fc (approximately 56kD) in 2 µl of plasma collected at day 2 from mice treated daily with 40 µg of TREM1-Fc. Anti-mouse IgG was used as endogenous control. ( C ) Q-PCR for different inflammatory transcripts (left) or pro-fibrotic transcripts, Collagen1a1 (Col1a1) and alpha smooth muscle actin ( Acta2 ), from whole kidney day 5 after U-IRI. ( D ) Representative images (left) and quantitative graphs (right) showing+F4/80 cells (green),+αSMA (red) or collagen deposition (Sirius Red staining) day 5 after U-IRI. (*P<0.05, n = 5–7/group, 3 independent experiments; Bar marker = 50 µm; Q-PCR results were normalized to sham +hIgG).

Article Snippet: To activate TREM-1 in BMDMφ, anti-TREM-1 antibodies (R&D) at 1 or 2 μg/ml or isotype IgG control (R&D) were applied.

Techniques: Control, Western Blot, Clinical Proteomics, Staining, Marker