A unique subset of CD11c-positive dendritic cells mediate immunological tolerance via TLR9 in Concanavalin A-induced liver injury in mice
Nobuhiro Nakamoto1, Hirotoshi Ebinuma1, Takanori Kanai1, Yuko Wakayama1, Nobuhito Taniki1, Hiroko Murata1, Yohei Mikami1, Po-sung Chu1, Kazuo Sugiyama1, Hidetsugu Saito2,1, Toshifumi Hibi1;
1Department of Internal Medicine, University of Keio, School of Medicine, Tokyo, Japan; 2 Department of Pharmacotherapeutics, Keio Unversity, School of Pharmacy, Tokyo, Japan
BACKGROUND & AIMS: We recently reported that C-C chemokine receptor (CCR) 9+ CD11 b+ macrophages play a critical role in the pathogenesis of acute liver injury by a single injection of Concanavalin A (Con A) in mice (Gastroenterology 2012). On the other hand, it is known that a repetitive injection of Con A results in immunological tolerance under a specific condition. In this study, we tried to elucidate a participation of a unique subset of APCs in mediating liver tolerance, especially focusing on the disease specific toll like receptors (TLRs). METHODS: Male C57BL/6 mice were re-injected with a sublethal dose of Con A 1, 3, or 7 days after an initial Con A administration to induce immunological tolerance. Liver mononuclear cells were separated 12 hours after the final Con A administration, and soluble cytokines production from these cells stimulated with TLR1-9 agonist in vitro was measured by Cytometric Bead Array (CBA) assay. RESULTS: Con A pretreated mice were partially protected from liver injury by Con A rechallenge 7 days after pretreatment, while liver damage was more aggravated by Con A rechallenge as early as 3 days after pretreatment. We next performed a serial analysis of the number and the function of hepatic APCs following Con A administration. The number of CD11 b+ F4/80+ macrophages dramatically increased that peaked at 24 hours and decreased thereafter. In contrast, the number of CD 11c+ DCs decreased that peaked at 24 hours perhaps due to an increased cell death, and returned to the baseline by 7 days. CD11c+ DCs within Con A-treated liver were phenotypically mature with increased expression of CD80, CD86, and MHC class2. In vitro stimulation of whole liver cells with TLR4, 6, and 9 agonist induced the production of pro-inflammatory cytokines such as IL-6 and TNF-α, while the production of IL-10 was only induced by TLR9 agonist stimulation and the Th 1 /Th2 balance via TLR9 shifted to Th2 dominant with time following Con A administration. Hepatic CD11c+ DCs sorted by MACS beads 7 days after Con A administration (CD11c+ DCs-D7) have a maximum potential to produce IL-10 and TGF-β by TLR9 agonist stimulation compared with other APCs subset. These CD11c+ DCs prompted naïve CD4 T cells to differentiate to a regulatory phenotype in the presence of TLR9 agonist in vitro. Finally, Pretreatment with CD11c+ DCs-D7, but not CD11c+ DCs sorted at earlier time point or CD11 b+ macrophages, protected mice from Con A induced acute liver damage in vivo. CONCLUSIONS: In summary, IL-10 producing MHC class2+ CD11c+ DCs play a role in mediating liver tolerance via TLR9 as an important negative regulator of the excessive liver inflammation by Con A in mice.
Toshifumi Hibi - Grant/Research Support: Abbott Japan, Ajinomoto Pharma, Astrazeneca Phramaceuticals, Janssen Pharmaceutical K.K, Tanabe Mitsubishi Pharma
The following people have nothing to disclose: Nobuhiro Nakamoto, Hirotoshi Ebinuma, Takanori Kanai, Yuko Wakayama, Nobuhito Taniki, Hiroko Murata, Yohei Mikami, Po-sung Chu, Kazuo Sugiyama, Hidetsugu Saito