Irisin inhibits neutrophil extracellular traps formation and protects against acute pancreatitis in mice

Abstract

Introduction: Irisin is a newly discovered myokine which links exercise to inflammation and inflammation-related diseases through macrophage regulation. However, the effect of irisin on the activity of inflammation related immune cells (such as neutrophils) has not been clearly described.

Objectives: The objective of our study was to explore the effect of irisin on the neutrophil extracellular traps (NETs) formation.

Methods: Phorbol-12-myristate-13-acetate (PMA) was used to construct a classic neutrophil inflammation model that was used to observe the formation of NETs in vitro. We studied the effect of irisin on NETs formation and its regulation mechanism. Subsequently, acute pancreatitis (AP) was used to verify the protective effect of irisin in vivo, which was an acute aseptic inflammatory response disease model closely related to NETs.

Results: Our study found that addition of irisin significantly reduced the formation of NETs via regulation of the P38/MAPK pathway through integrin αVβ5, which might be the one of key pathways in NETs formation, and which could theoretically offset the immunoregulatory effect of irisin. Systemic treatment with irisin reduced the severity of tissue damage common in the disease and inhibited the formation of NETs in pancreatic necrotic tissue of two classical AP mouse models.

Conclusion: The findings confirmed for the first time that irisin could inhibit NETs formation and protect mice from pancreatic injury, which further elucidated the protective effect of exercise on acute inflammatory injury.

Keywords: Acute pancreatitis; Irisin; Neutrophil extracellular traps; Neutrophils; p38.

Graphical abstract

Fig. 1

Irisin inhibits NETs formation. Bone marrow neutrophils isolated from six to eight-weeks-old C57BL/6J male mice are cultured and stimulated with PMA (100 nM), and incubated with different doses of irisin (5, 10, and 20 ng/ml), then stained for flow cytometry analysis. Representative flow cytometry gating of bone marrow neutrophils of C57BL/6J mice (CD45.2⁺CD11b⁺Ly6G⁺). (A) Representative flow cytometry plots and bar graphs depicting the proportion of neutrophils and the expression levels of MPO and ROS. Data is presented as mean ± SE (N = 4 for each group). (B) Representative immunofluorescence image of CitH3 in magnification at 400× and 1000× (N = 6 for each group). Scale Bar = 50 μM. (C) Densitometric analysis of CitH3 fluorescence (N = 6 for each group). (D) The supernatant levels of IL-1β, IL-6, TNF-α, and MCP-1 are detected using ELISA. (N = 6 for each group). (E) Protein levels of PAD4 in neutrophils analyze using Western blotting (N = 3 for each group). (F) Relative protein expression of PAD4 (N = 3 for each group); β-actin is used as control for protein loading.

Fig. 2

Irisin restrains NETs formation depending on P38/MAPK signaling pathway. (A) KEGG enrichment analysis of the overlapping genes of the differential genes of PMA vs. NC and PMA + irisin vs. PMA. (B) Western blot analysis of protein levels of p-p38, p-Erk, p38, and Erk in neutrophils (N = 3 for each group). (C) Bone marrow-derived neutrophils were first incubated with SB203580 for 30 min, followed by treatment with irisin for 30 min, and by PMA stimulation for 4 h. Representative flow cytometry gating of neutrophils (CD45.2⁺CD11b⁺Ly6G⁺). Representative flow cytometry plots and bar graphs depicting the proportion of neutrophils and the expression levels of MPO and ROS. Data is presented as mean ± SE (N = 4 for each group). (D) Representative immunofluorescence image of CitH3 at magnifications 400× and 1000×, and densitometric analysis of CitH3 fluorescence (N = 6 per group). Scale Bar = 50 μM. (E) The supernatant levels of IL-1β, IL-6, TNF-α and MCP-1 are measured using ELISA (N = 6 per group).

Fig. 3

Integrin αVβ5 suppresses NETs formation. (A) Western blot analysis of integrin αVβ5 level in neutrophils (N = 4 per group). (B) Relative protein expression of integrin αVβ5; β-actin is used as a control for protein loading (N = 4 per group). (C) Representative flow cytometry gating of bone marrow neutrophils of C57BL/6J mice (CD45.2⁺CD11b⁺Ly6G⁺). Representative flow cytometry plots and bar graphs depicting the proportion of neutrophils and the expression levels of MPO and ROS. Data is presented as mean ± SE (N = 4 for each group). (D) Representative immunofluorescence image of CitH3 in magnification 400× and 1000× (N = 6 for each group). Scar Bar = 50 μM. (E) Densitometric analysis of CitH3 fluorescence (N = 6 for each group). (F) Western blot analysis of PAD4 levels in neutrophils. Relative protein expression of PAD4; β-actin is used as a control for protein loading (N = 3 each group).

Fig. 4

Irisin represses NETs formation via the integrin αVβ5-p38/MAPK signaling pathway. (A) Western blot analysis of protein levels of p-p38 and p38 in neutrophils. Relative protein expression of p-P38, p38 is used as controls for protein loading (N = 4 per group). (B–C) Representative flow cytometry gating of bone marrow neutrophils of C57BL/6J mice (CD45.2⁺CD11b⁺Ly6G⁺). Representative flow cytometry plots and bar graphs depicting the proportion of neutrophils and the expression levels of MPO and ROS. Data is presented as mean ± SE (N = 4 for each group). (D) Representative immunofluorescence image of CitH3 in magnification 400× and 1000× (N = 6 for each group). Scar Bar = 50 μM. (E) Densitometric analysis of CitH3 fluorescence (N = 6 for each group).

Fig. 5

Irisin curbs NETs formation and alleviates disease severity in MAP mice model. (A) Representative HE staining of pancreatic tissues at magnifications 100× and 400×. Scale Bar = 50 μM. (B) Serum levels of amylase and lipase. (C–D) The pathological scores of pancreatic tissues. (E) Representative immunofluorescence image of CitH3 and MPO at magnifications 400× and 1000×. Scale Bar = 50 μM. (F) Densitometric analysis of CitH3 and MPO fluorescence. (N = 7 per group).

Fig. 6

Integrin αVβ5 inhibitor inhibits NETs formation and mitigates disease severity in MAP mice model. (A) Representative HE staining of pancreatic tissues at magnifications 100× and 400×. Scale Bar = 50 μM. (B) Serum levels of amylase and lipase. (C–D) The pathological scores of pancreatic tissues. (E) Representative immunofluorescence image of CitH3 and MPO at magnifications 400× and 1000×. Scale Bar = 50 μM. (F) Densitometric analysis of CitH3 and MPO fluorescence (N = 7 per group).