Extracellular CIRP as an endogenous TREM-1 ligand to fuel inflammation in sepsis
Naomi-Liza Denning, Monowar Aziz, Atsushi Murao, Steven D. Gurien, Mahendar Ochani, Jose M. Prince, Ping Wang
Naomi-Liza Denning, Monowar Aziz, Atsushi Murao, Steven D. Gurien, Mahendar Ochani, Jose M. Prince, Ping Wang
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Research Article Immunology Inflammation

Extracellular CIRP as an endogenous TREM-1 ligand to fuel inflammation in sepsis

Abstract

Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently discovered damage-associated molecular pattern. Understanding the precise mechanism by which it exacerbates inflammation is essential. Here we identified that eCIRP is a new biologically active endogenous ligand of triggering receptor expressed on myeloid cells-1 (TREM-1), fueling inflammation in sepsis. Surface plasmon resonance revealed a strong binding affinity between eCIRP and TREM-1, and fluorescence resonance energy transfer assay confirmed eCIRP’s interaction with TREM-1 in macrophages. Targeting TREM-1 by its siRNA or a decoy peptide, LP17, or by using TREM-1–/– mice dramatically reduced eCIRP-induced inflammation. We developed a potentially novel 7-aa peptide derived from human eCIRP, M3, which blocked the interaction of TREM-1 and eCIRP. M3 suppressed inflammation induced by eCIRP or agonist TREM-1 antibody cross-linking in murine macrophages or human peripheral blood monocytes. M3 also inhibited eCIRP-induced systemic inflammation and tissue injury. Treatment with M3 further protected mice from sepsis, improved acute lung injury, and increased survival. Thus, we have discovered a potentially novel TREM-1 ligand and developed a new peptide, M3, to block eCIRP–TREM-1 interaction and improve outcomes in sepsis.

Authors

Naomi-Liza Denning, Monowar Aziz, Atsushi Murao, Steven D. Gurien, Mahendar Ochani, Jose M. Prince, Ping Wang

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Figure 4

M3, a small CIRP-derived peptide, inhibits eCIRP and TREM-1 interaction.

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M3, a small CIRP-derived peptide, inhibits eCIRP and TREM-1 interaction....
(A) Partial aa sequence of CIRP highlighting an area of similarity with PGLYRP1. RAW264.7 cells were treated with 10 μg/mL of peptides M1, M2, or M3 for 30 minutes, then stimulated with rmCIRP (1 μg/mL). After 24 hours, TNF-α in culture supernatants was measured. Data are expressed as mean ± SEM obtained from 2 independent experiments (n = 5–7 wells/peptide group) and were compared by 1-way ANOVA and Tukey’s method (*P < 0.05 vs. unstimulated cells, and #P < 0.05 vs. rmCIRP-treated cells). (B) SPR between rmTREM-1 and M3. (C) RAW264.7 cells and (D) primary peritoneal macrophages were treated with M3 or M3-Sc1 (both 10 μg/mL) for 30 minutes. Cells were then stimulated with PBS or rmCIRP (5 μg/mL), and FRET analysis was performed as described in Figure 1C. Data are expressed as mean ± SEM obtained from 3 independent experiments; n = 7–9/group, compared by 1-way ANOVA and Tukey’s method (*P < 0.05 vs. CD11b + rmCIRP, and #P < 0.05 vs. TREM-1 + rmCIRP). (E) To activate RAW264.7 cells through TREM-1, 96-well plates were precoated with 20 μg/mL of an agonist anti–TREM-1 mAb. Then, 5 × 104 cells/well were premixed with PBS control or M3 (10 μg/mL) or scramble peptide (10 μg/mL) for 30 minutes, then plated. TNF-α production was measured in the culture supernatants after an additional 24 hours of incubation. The experiment was performed 2 independent times with n = 5–6 wells per group. Scramble peptide groups, n = 5/group. Data are expressed as mean ± SEM and were compared by 1-way ANOVA and Tukey’s method (*P < 0.05 vs. uncoated; #P < 0.05 vs. TREM-1 Ab + PBS). (F and G) RAW264.7 cells were treated with M3 or scramble peptide for 30 minutes, then stimulated with PBS or rmCIRP (1 μg/mL). After 24 hours, (F) TNF-α and (G) IL-6 in culture supernatants were assessed. Data are expressed as mean ± SEM obtained from 3 independent experiments and compared by 1-way ANOVA and Tukey’s method (*P < 0.05 vs. PBS-treated cells; #P < 0.05 vs. rmCIRP + PBS). (H) Human macrophages were treated with M3 for 30 minutes, then stimulated with PBS or rmCIRP (1 μg/mL). After 24 hours, TNF-α in culture supernatants was measured. Data are expressed as mean ± SEM (n = 5 wells/group) and were compared by Kruskal-Wallis test with Dunn’s method (*P < 0.05 vs. PBS-treated cells; #P < 0.05 vs. rmCIRP + PBS). Ab, antibody.