L161982 alleviates collagen-induced arthritis in mice by increasing Treg cells and down-regulating Interleukin-17 and monocyte-chemoattractant protein-1 levels

Abstract

Background: To investigate the effects and potential mechanism of L161982 (a kind of EP4 antagonist) on the collagen-induced arthritis (CIA) mice model.

Methods: The CIA mice model were first established by immunizing with Chicken Type II Collagen on DBA/1 mice. The CIA groups were administered once a day for 2 weeks with either 5 mg/kg L161982 by intraperitoneal injections (IP), 200 U celecoxib by intragastrical injections, or 100 μl PBS (IP). At the end of the study, total arthritis score and histopathologic examination were assessed to determine CIA severity. The plasma and tissue expressions of IL-17 and monocyte chemoattractant protein-1 (MCP-1) were detected by enzyme-linked immunosorbent assay (ELISA) and Immunohistochemical staining (IHC) respectively; The number of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Treg) determined as a proportion of total CD4⁺ cells in the lymph nodes and spleen. We also tested the proliferation of isolated Tregs and the ratio of Th17 polarization of Naïve T cells under the treatment of L161982 by BrdU assay and flow cytometry respectively.

Results: CIA mice treated with L161982 showed reduced arthritis scores, joint swellings, cracked cartilage surface, and less hyperplasia in the connective tissue of the articular cavity. Plasma and tissue IL-17 and MCP-1 decreased, while the proportion of Treg cells is increased both in the spleen and lymph nodes of CIA mice. Otherwise, L161982 have no direct effect on Tregs proliferation; a decreased tendency of Th17 polarization in vitro were observed in L161982-treated naïve T cells.

Conclusion: Although less effective than Celecoxib, L161982 also resulted in a reduction of ankle joint inflammation in CIA mice. L161982 reduces the RA severity in CIA mice through inhibition of IL-17 and MCP-1, increasing Treg cells, and reducing inflammation. The mechanism of the reduction of IL-17 in plasma or tissue after administration of L161982 might be potentially derived from the suppression of CD4⁺ T cells differentiation into Th-17 cells.

Keywords: Collagen-induced arthritis; EP4 antagonist; Interleukin-17; Monocyte chemotactic protein-1; Rheumatoid arthritis.

Fig. 1

Macroscopic observations of the redness and swelling of toes 35 days after the first immunization. Upper: Time and immuniaztion of the animal experiment. Bottom: comparing with model-control group, CIA-model groups presented all redness and swelling inferior the ankle joint; Within CIA-model groups, L161982 and celecoxib group just showed less red and swollen than blank and PBS groups. The number in the upright of each figure means the arthritis score (AS) of the mouse

Fig. 2

Histological examination of mice hind paws. Blank CIA mice and PBS treated CIA mice showed hyperplasia of the synovial tissue, increased new blood vessels. Increased inflammatory cells infiltration, and cracked and denudated cartilage. Celecoxib and L161982 treated CIA mice showed less hyperplasia and inflammatory cells infiltration. Synovial hyperplasia in the articular cavity is marked in black arrows. Small maps (Magnification 50 μm) within each larger picture (Magnification 200 μm) highlight areas indicated by arrows. Slides were hematoxylin and eosin stained and magnified at 200 μm/50 μm)

Fig. 3

Immunohistochemical staining of CIA-model mice for testing the tissue expression of MCP-1,IL-17 and C-caspase 3. Celecoxib and L161982 treated CIA mice showed lower level of MCP-1 and IL-17 than the blank and PBS group. The expression of C-caspase 3 in all groups were looked no difference. “N.C” i.e. negative control, means tissue were stained without the corresponding primary antibody. C-caspase 3 means cleaved caspase 3

Fig. 4

The effects of L-161982 on Treg cells and Th17cells. Fig 4a, Flow cytometry analysis showed as well as celecoxib, L-161982 increase the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells both in spleen and lymph node of the CIA-model mice; Fig. 4b, L161982 could not affect the proliferation of Treg cells in vitro. Purified CD4⁺CD25⁺ cells were stimulated with anti-CD3, anti-CD28 and PEG2 with or without L161982, then cells were mixed with BrdU for proliferation analysis as described in mothed; Fig. 4c, Naïve T cells were isolated from mouse spleen and stimulated with cytokines for differentiating into Th17 cells. The ratio of Th17 cells were decreased by treating with L161982. Additional file 1