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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.

SUBMITTER: Denning NL

PROVIDER: S-EPMC7141396 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Denning Naomi-Liza NL Aziz Monowar M Murao Atsushi A Gurien Steven D SD Ochani Mahendar M Prince Jose M JM Wang Ping P

JCI insight 20200312 5

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 tr ...[more]

PMID: 32027618

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Project description:BackgroundIn sepsis, intestinal barrier dysfunction is often caused by the uncontrolled death of intestinal epithelial cells (IECs). CD4CD8αα intraepithelial lymphocytes (IELs), a subtype of CD4+ T cells residing within the intestinal epithelium, exert cytotoxicity by producing granzyme B (GrB) and perforin (Prf). Extracellular cold-inducible RNA-binding protein (eCIRP) is a recently identified alarmin which stimulates TLR4 on immune cells to induce proinflammatory responses. Here, we hypothesized that eCIRP enhances CD4CD8αα IEL cytotoxicity and induces IEC death in sepsis.MethodsWe subjected wild-type (WT) and CIRP-/- mice to sepsis by cecal ligation and puncture (CLP) and collected the small intestines to isolate IELs. The expression of GrB and Prf in CD4CD8αα IELs was assessed by flow cytometry. IELs isolated from WT and TLR4-/- mice were challenged with recombinant mouse CIRP (eCIRP) and assessed the expression of GrB and Prf in CD4CD8αα by flow cytometry. Organoid-derived IECs were co-cultured with eCIRP-treated CD4CD8αα cells in the presence/absence of GrB and Prf inhibitors and assessed IEC death by flow cytometry.ResultsWe found a significant increase in the expression of GrB and Prf in CD4CD8αα IELs of septic mice compared to sham mice. We found that GrB and Prf levels in CD4CD8αα IELs were increased in the small intestines of WT septic mice, while CD4CD8αα IELs of CIRP-/- mice did not show an increase in those cytotoxic granules after sepsis. We found that eCIRP upregulated GrB and Prf in CD4CD8αα IELs isolated from WT mice but not from TLR4-/- mice. Furthermore, we also revealed that eCIRP-treated CD4CD8αα cells induced organoid-derived IEC death, which was mitigated by GrB and Prf inhibitors. Finally, histological analysis of septic mice revealed that CIRP-/- mice were protected from tissue injury and cell death in the small intestines compared to WT mice.ConclusionIn sepsis, the cytotoxicity initiated by the eCIRP/TLR4 axis in CD4CD8αα IELs is associated with intestinal epithelial cell (IEC) death, which could lead to gut injury.

Project description:BackgroundSepsis is a life-threatening disease syndrome caused by a dysregulated host response to infection and injury. Extracellular cold-inducible RNA-binding protein (eCIRP) acts as a damage-associated molecular pattern. Peritoneal cavity (PerC) B-1a cells attenuate inflammation and tissue injury by spontaneous releasing natural IgM and IL-10. Sialic acid-binding immunoglobulin-type lectin-G (Siglec-G) is a CD33-related receptor highly expressed in B-1a cells to serve critical immunoregulatory functions. In sepsis, B-1a cell numbers in PerC are decreased. We hypothesized that eCIRP causes the reduction of PerC B-1a cells and alters their function during sepsis.MethodsSepsis was induced in WT and CIRP-/- mice by cecal ligation and puncture (CLP). PerC washout cells were collected and B-1a cells and Siglec-G were assessed by flow cytometry. Mice were i.p. injected with recombinant murine (rm) CIRP and after 20 h, Siglec-G expression in PerC B-1a cells were assessed. PerC B-1a cells were treated with rmCIRP for 4 h and Siglec-G expression was assessed. PerC B-1a cells were pre-treated with anti-Siglec-G Ab and then after stimulated with rmCIRP for 24 h, IL-6 levels in the culture supernatants were assessed.ResultseCIRP levels in the PerC were elevated in septic mice. In WT mice, the frequencies and numbers of total and Siglec-G+ B-1a cells in the PerC were significantly decreased in the CLP group compared to sham group, whereas in CIRP-/- mice, their frequencies and numbers in sepsis were significantly rescued compared to WT septic mice. Mice injected with rmCIRP showed decreased frequencies and numbers of total and Siglec-G+ PerC B-1a cells compared to PBS-injected mice. In vitro treatment of PerC B-1a cells with rmCIRP demonstrated significant reduction in Siglec-G mRNA and protein compared to PBS group. PerC B-1a cells treated with anti-Siglec-G Ab had significantly higher production of IL-6 in response to rmCIRP compared to IgG control. Anti-Siglec-G Ab treated B-1a cells co-cultured with macrophages produced significantly higher levels of IL-6, and TNF-α, and lower levels of IL-10 compared to IgG-treated B-1a cells and macrophage co-cultures stimulated with rmCIRP.ConclusioneCIRP reduces PerC B-1a cell pool and skews them to a pro-inflammatory phenotype by downregulating Siglec-G expression. Targeting eCIRP will retain Siglec-G expressing B-1a cells in the PerC and preserve their anti-inflammatory function in sepsis.

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

Publications

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

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