Date Published: August 8, 2018
Publisher: Public Library of Science
Author(s): Miri Blank, Tomer Bashi, Jordan Lachnish, Dana Ben-Ami-Shor, Ora Shovman, Mati Fridkin, Miriam Eisenstein, Alexander Volkov, Iris Barshack, Yehuda Shoenfeld, Dominique Heymann.
A novel small molecule named tuftsin-phosphorylcholine (TPC), which is linked to the biological activity of helminths, was constructed. The current study address the effect of TPC treatment in established collagen-induced arthritis (CIA) mice and propose TPC bi-functional activity. TPC treatment was initiated when clinical score was 2 to 4. Arthritis scores in TPC treated mice were lower compared to mice treated with vehicle (P < 0.001). Joint staining showed normal joint structure in TPC-treated mice compared to control groups treated with phosphate buffered saline (PBS), phosphorylcholine, or tuftsin, which exhibited severely inflamed joints. TPC enhanced anti-inflammatory response due to increased IL-10 secretion, and reduced pro-inflammatory cytokine secretion (IL-1-β, IL-6, TNF-αP < 0.001). Furthermore, TPC therapy increased expansion of CD4+CD25+FOXP3+T regulatory cells and IL-10+CD5+CD1d+B regulatory cells. We propose that the immunomodulatory activity of TPC can be a result of a bi-specific activity of TPC: (a) The tuftsin part of the TPC shifts RAW macrophage cells from pro-inflammatory macrophages M1 to anti-inflammatory M2-secreting IL-10 (P < 0.001) through neuropilin-1 and (b) TPC significantly reduce mouse TLR4 expression via NFkB pathway by HEKTM cells (P < 0.02) via the phosphorylcholine site of the molecule. Our results indicate that TPC, significantly ameliorated established CIA by its immunomodulatory activity. These data could lead to a novel self bi-functional small molecule for treating patients with progressive RA.
Rheumatoid arthritis (RA) is a chronic, systemic autoinflammatory disease. It usually manifests as stiffness, pain, and swelling of the joints . Similar to other autoimmune diseases, RA has a genetic background associated mainly with HLA-DRB1, PTPN22, and TRAF1-C5 [2,3]. Environmental factors also contribute to the development of RA; the most prominent of these are smoking and infections, such as Epstein-Barr virus (EBV) infection [4,5]. One of the main explanations for the joint inflammation is the presence of citrullinated proteins followed by production of antibodies targeting these proteins (anti-citrullinated protein antibodies [ACPAs]) . In RA patients, the function of T regulatory (Treg) cells is impaired and increases Treg cellscount is well-correlated with a better clinical response in patients and animal models [7,8]. Furthermore, several studies of cells derived from RA patients indicated that the number of B10 regulatory (Breg) cells was inversely correlated with RA severity . Impaired Breg activity and high levels of IFN-γ expressing cells suppressed Treg cell differentiation and worsened arthritis in a murine model .
A decade ago the phosporylcholine moiety of the filarial nematode was found to be an immunomodulator in a host microenvioroment . We conjugated phosphorylcholine to a self immunomodulatory molecule, tuftsin, and named this small molecule—TPC. Tuftsin, having the sequence Thr-Lys-Pro-Arg, naturally occurs in human blood. This peptide is a fragment of the heavy chain Fc (289–292) of an IgG. Tuftsin is an endogenous immunomodulator of a wide spectrum of biological activity, It enhances phagocytosis, chemotaxis and pinocytosis and has antimicrobial and anticoagulant properties. Tuftsin targets FcγR and neuropilin-1. It is used as an adjuvant for mucosal vaccine based on HE-ORF2 and HA-VP1 (Hepatitis E virus and Hepatitis A virus) [22, 31].
This study was designed to assess the effect of helminth-derived phosphorylcholine conjugated to the self immunomodulatory molecule tuftsin, TPC, treatment on an established CIA. We have succeeded to show that TPC inhibits the clinical score of disease and the inflammatory process in the joints, as illustrated by clinical arthritis scoring system and histological assessment. The process of TPC immunomodulatory activity was illustrated by its ability to inhibit the secretion of pro-inflammatory cytokines, such as TNF-α, IL-1-β, and IL-6, and to upregulate IL-10 expression. The IL-10 source was Treg and Breg expansion as well as a possible conversion of macrophages to anti-inflammatory M2 macrophages secreting IL-10 via neuropilin-1. The amelioration of CIA was attributed to the bi-functional activity of TPC, targeting TLR4 through the phosphorylcholine leading to NF-kB inhibition and targeting neuropilin-1 via the tuftsin end of the molecule, leading to macrophages shift towards M2 anti-inflammatory secreting IL-10. The bi-functional activity resulted the anti-inflammatory network scenario. The molecular basis for all these functions should be further analyzed.