Background/Purpose: Siglecs (Sialic acid-binding immunoglobulin-type lectins) are type1 transmembrane proteins and expressed on cell surface of various immunocytes. Siglec9 is a member of CD33 related Siglecs which down-regulate both innate and acquired immune response. In our earlier studies, soluble Siglec9 was identified in the culture supernatant fluid of pulpal cells as a powerful anti-inflammatory element and has been found to be effective in several inflammatory diseases. The aim of this study is to investigate the efficacy of Siglec9 on murine collagen induced arthritis (CIA) model in vivo and the activation of cultured murine macrophage in vitro. 

Methods: To initiate CIA model, DBA/1J mice were immunized subcutaneously with bovine type II collagen emulsified with adjuvant. Recombinant Siglec9 (5 or 50ng/gm body weight) was administered intravenously to the mice with CIA weekly from day 23 (primary immunization in day 0) to day 42. The arthritis score and the histologic score were evaluated using the parameters described in a previous report. The serum concentration of TNFα was measured using an ELISA assay. In vitro, murine macrophage cell-line RAW264.7 was cultured in monolayers, stimulated with 10 ng/ml of IFNγ, and concurrently treated with Siglec9 (0–10ng/ml) for 12 hours. Total RNA was extracted and subjected to real-time polymerase chain reaction (PCR) analysis to determine the mRNA expression of TNFα, IL-6, iNOS, as M1 markers, and CD206, Arginage-1, as M2 markers and GAPDH for RAW264.7. The effect of Siglec9 on the expression levels of TNFα, IL-6, and iNOS protein in RAW264.7 was evaluated by ELISA and Western blot analysis. To clarify whether sialic acid are necessary for Siglec9 to demonstrate inhibitory effect, we used sialidase to remove sialic acid. RAW264.7 was pre-incubated with sialidase for 1 h before stimulation protocols were administered.

Results: Treatment with Siglec9 in CIA mice significantly suppressed the incidence rate and severity of arthritis (based on the arthritis score) in a dose-dependent manner. The beneficial effect was maximal in mice treated with 50ng/gm of Siglec9. Serum TNFα level was significantly inhibited in groups treated with Siglec9 compared with control group. Treatment with Siglec9 significantly reduced the histologic score in both knee and ankle joints. RT–PCR analysis revealed that treatment with Siglec9 decreased the mRNA expression of TFNα, IL-6, and iNOS in RAW264.7 stimulated with IFNγ in a dose-dependent manner. However, we could not find significant increase in mRNA expression of M2 marker such as CD206, arginaze-1, fizz1 by treatment with Siglec9. The protein expression of each M1 markers were also suppressed by treatment with Siglec9. Sialidase treatment canceled the inhibitory effect which was seen in treatment group with Siglec9.

Conclusion: Siglec9 reduced disease activity of arthritis and suppressed histological inflammation and joint destruction, and in vitro, M1 markers (TNFα, IL-6, iNOS) were remarkably suppressed by treatment with Siglec9 in dose dependent manner. The inhibitory effect of Siglec9 was canceled by removing sialic acid with sialidase.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/siglec9-suppresses-arthritis-in-collagen-induced-mice-model-and-inhibits-m1-activation-of-raw264-7-macrophages/