Soluble Fibrinogen-Like Protein 2 Downregulation and Th17/Treg Imbalance in a Taurocholate-Induced Murine Experimental Model of Severe Acute Pancreatitis

Abstract

Background: Severe acute pancreatitis (SAP) is associated with tremendous systemic inflammation, T-helper 17 (Th17) cells, and regulatory T (Treg) cells play an essential role in the inflammatory responses. Meanwhile, soluble fibrinogen-like protein 2 (Sfgl2) is a critical immunosuppressive effector cytokine of Treg cells and modulates immune responses. However, the impact of SAP induction on Sfgl2 expression and the role of Sfgl2 in immunomodulation under SAP conditions are largely unknown.

Methods: A taurocholate-induced mouse SAP model was established. The ratios of CD4⁺CD25⁺Foxp3⁺ Treg cells or CD4⁺IL-17⁺ Th17 cells in blood and pancreatic tissues as well as surface expression of CD80, CD86, and major histocompatibility complex class II (MHC-II) were determined by flow cytometry. Gene mRNA expression was determined by qPCR. Serum amylase and soluble factors were quantitated by commercial kits. Bone marrow-derived dendritic cells (DCs) were generated, and NF-κB/p65 translocation was measured by immunofluorescence staining.

Results: SAP induction in mice decreased the Th17/Treg ratio in the pancreatic tissue and increased the Th17/Treg ratio in the peripheral blood. In addition, SAP was associated with a reduced level of Sfgl2 in the pancreatic tissue and blood: higher levels of serum IL-17, IL-2, IFN-α, and TNF-α, and lower levels of serum IL-4 and IL-10. Furthermore, the SAP-induced reduction in Sfgl2 expression was accompanied by dysregulated maturation of bone marrow-derived DCs.

Conclusions: SAP causes reduced Sfgl2 expression and Th17/Treg imbalance, thus providing critical insights for the development of Sfgl2- and Th17/Treg balance-targeted immunotherapies for patients with SAP.

Keywords: Th17/Treg imbalance; T‐helper 17 cell; immunomodulation; inflammation; regulatory T cell; severe acute pancreatitis; soluble fibrinogen–like protein 2.

FIGURE 1

Histological and cellular analyses confirmed that sodium taurocholate administration induced SAP in mice. (A,B) After anesthesia, the mice were divided into two groups: The sham operation (Sham) group and the SAP induction (SAP) group. The SAP mice were retrogradely injected with sodium taurocholate (5%; 1 mL/kg body weight) into the biliopancreatic duct, and the sham mice underwent surgery without sodium taurocholate infusion. At 4 h after the SAP induction or sham operation, the mice were anesthetized and euthanized. The pancreatic tissues were harvested immediately for histological analysis and dissociated for cellular analysis. (A) Representative images showing the histological changes in pancreatic tissues by H&E staining. Magnification, 400×. (B) Representative images showing the cellular morphological changes in dissociated pancreatic cells. Magnification, 20×.

FIGURE 2

SAP induction changed the frequencies of Treg cells and Th17 cells in mouse peripheral blood. (A–D) At 4 h after surgery, the frequencies of CD25⁺Foxp3⁺ Treg cells and CD4⁺IL‐17⁺ Th17 cells among the total CD4⁺ T cells in blood samples from the Sham mice and SAP mice were measured by flow cytometry. Representative profiles showing the percentages of CD25⁺Foxp3⁺ Treg cells (A) and CD4⁺ IL‐17⁺ Th17 cells (C) in blood samples from the Sham and SAP groups. The percentages of blood Treg cells (B) and Th17 cells (D) are summarized. n = 5 mice per group, *p < 0.05, between the indicated groups.

FIGURE 3

SAP induction changed the frequencies of Treg cells and Th17 cells in mouse pancreatic tissues. (A–D) At 4 h after surgery, the frequencies of CD25⁺Foxp3⁺ Treg cells and CD4⁺IL‐17⁺ Th17 cells among the total CD4⁺ T cells in pancreatic tissues from the sham mice and SAP mice were measured by flow cytometry. Representative profiles showing the percentages of CD25⁺Foxp3⁺ Treg cells (A) and CD4⁺ IL‐17⁺ Th17 cells (C) in pancreatic tissues samples from the Sham and SAP groups. The percentages of Treg cells (B) and Th17 cells (D) in the pancreatic tissues are summarized. (E, F) The mRNA levels of Foxp3 (E) and Sfgl2 (F) in the pancreatic tissues were quantitated by qPCR. n = 5 mice per group, *p < 0.05, between the indicated groups.

FIGURE 4

SAP induction lowered the serum Sfgl2 level and changed the levels of soluble inflammatory mediators in mouse blood. (A–H) At 4 h after surgery, the relative serum levels of amylase (A), Sfgl2 (B), IL‐2 (C), IL‐4 (D), IL‐10 (E), IL‐17 (F), IFN‐α (G), and TNF‐α (H) were measured and compared between the Sham group and the SAP group. n = 5 mice per group, *p < 0.05, between the indicated groups.

FIGURE 5

SAP induction in mice changed the NF‐κB/p65 nuclear translocation and activation status of bone marrow–derived DCs. (A) Bone marrow cells were isolated from mice at 4 h after surgery, and the bone marrow–derived DCs were enriched and purified after induction in culture medium containing IL‐4 and GM‐CSF. Representative images show the cellular morphology of DCs in the Sham and SAP groups. Magnification, 20×. (B) Representative images of immunofluorescence staining show the changes in nuclear translocation of NF‐κB‐p65 in the bone marrow–derived DCs from the Sham and SAP groups. Magnification, 20×. (C–E) The relative surface levels (percentages of positively stained cells among the total DCs) of DC activation markers including CD80 (C), CD86 (D), and MHC‐II (E) on bone marrow–derived DCs from the Sham and SAP groups were compared. n = 5 mice per group, *p < 0.05, between the indicated groups.