The Role of Nucleotide-binding Oligomerization Domain 2 on the Immunomodulatory Function of Mesenchymal Stem Cells in Mouse Colitis Model

Abstract

Mesenchymal stem cells (MSCs) are multipotent adult stromal cells that can self-renew and differentiate into various cell types of mesodermal lineage. Moreover, MSCs are recently known to possess regulatory function on immune cells which makes them a promising tool for the treatment of inflammatory and autoimmune diseases. The interaction between MSCs and immune cells through soluble factors and adhesion molecules has been reported to be crucial for the immunomodulatory effect of MSCs. Innate immune receptors, such as Toll-like receptors (TLRs) and Nod-like receptors (NLRs), are known to trigger an initial immune responses against microbial infection. Although studies suggest that activation of TLRs modulate the function of mesenchymal stem cells (MSCs), little is known about the role of NLRs on the MSC function. In this study, I investigated whether Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) and 2 (NOD2), well-known receptors in NLR family, regulate the functions of human umbilical cord blood-derived MSCs (hUCB-MSCs). TLR2, TLR4, NOD1, NOD2 and receptor interacting protein 2 (RIP2), an adaptor protein of NOD1 and NOD2 were expressed in hUCB-MSCs. Stimulation with each agonist

Pam3CSK4 for TLR2, Lipopolysaccharide (LPS) for TLR4, L-Ala-gamma-D-Glu-meso- diamino-pimelic acid (Tri-DAP) for NOD1, and muramyl dipeptide (MDP) for NOD2, led to Interleukin (IL)-8 production in hUCB-MSC, suggesting that the expressed receptors are functional in hUCB-MSC. None of agonists influenced proliferation of hUCB-MSCs. I next examined whether TLR and NLR agonists affect the differentiation of hUCB-MSCs. Pam3CSK4 and Tri-DAP strongly enhanced osteogenic differentiation through the induction of ERK phosphorylation in hUCB-MSCs, and LPS and MDP also slightly augmented osteogenesis. Treatment of U0126 (MEK1/2 inhibitor) restored osteogenic differentiation enhanced by Pam3CSK4. Tri-DAP and MDP slightly inhibited adipogenic differentiation of hUCB-MSCs, but Pam3CSK4 and LPS did not. On chondrogenic differentiation, all TLR and NLR agonists could promote chondrogenesis of hUCB-MSCs (Kim et al., 2010). Decreased levels or function of NOD2 are associated with Crohns disease. NOD2 is known to regulate intestinal inflammation, and also is expressed by human umbilical cord blood-derived mesenchymal stem cells, to regulate their differentiation. I investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis. Colitis was induced in mice by administration of dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS). Mice then were given intraperitoneal injections of NOD2-activated hUCB-MSCs, and colon tissues and mesenteric lymph nodes were collected for histologic analyses. Administration of hUCB-MSCs reduced the severity of colitis in mice. The anti-inflammatory effects of hUCB-MSCs were greatly increased by activation of NOD2 by its ligand, MDP. Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin IL-10 and infiltration by T regulatory cells, and reduced production of inflammatory cytokines. A bromodeoxyuridine (BrdU) assay was used to determine the ability of hUCB-MSCs to inhibit proliferation of human mononuclear cells (hMNCs) in culture. Proliferation of mononuclear cells was inhibited significantly by co-culture with hUCB-MSCs that had been stimulated with MDP. MDP induced prolonged production of prostaglandin (PG)E2 in hUCB-MSCs via the NOD2–RIP2 pathway, which suppressed proliferation of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs in response to MDP increased production of IL-10 and T regulatory cells. In mice, production of PGE2 by MSCs and subsequent production of IL-10 were required to reduce the severity of colitis (Kim et al., 2013).

Taken together, these findings suggest that (1) NOD1 and NOD2 as well as TLRs are involved in regulating the differentiation of MSCs, and that (2) activation of NOD2 is required for the ability of hUCB-MSCs to reduce the severity of colitis in mice and NOD2 signaling increases the ability of these cells to suppress mononuclear cell proliferation by inducing the production of PGE2.