Tolerogenic Dendritic Cells Generated by In Vitro Treatment With SAHA Are Not Stable In Vivo.

The aim of this study is to examine whether the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can generate dendritic cells (DCs) with a stable tolerogenic phenotype to counteract autoimmune responses in an animal model of multiple sclerosis. We investigated if the tolerogenic potency of DCs could be increased by continuous treatment during in vitro differentiation toward DCs compared to standard 24-h in vitro treatment of already terminally differentiated DCs. We show that in vitro treatment with SAHA reduces the generation of new CD11c(+) DCs out of mouse bone marrow. SAHA-generated DCs show reduced antigen-presenting function as evidenced by a reduction in myelin endocytosis, a decreased MHC II expression, and a failure to upregulate costimulatory molecules upon LPS challenge. In addition, SAHA-generated DCs display a reduction in proinflammatory cytokines and molecules involved in apoptosis induction, inflammatory migration, and TLR signaling, and they are less immunostimulatory compared to untreated DCs. We demonstrated that the underlying mechanism involves a diminished STAT1 phosphorylation and was independent of STAT6 activation. Although in vitro results were promising, SAHA-generated DCs were not able to alleviate the development of experimental autoimmune encephalomyelitis in mice. In vitro washout experiments demonstrated that the tolerogenic phenotype of SAHA-treated DCs is reversible. Taken together, while SAHA potently boosts tolerogenic properties in DCs during the differentiation process in vitro, SAHA-generated DCs were unable to reduce autoimmunity in vivo. Our results imply that caution needs to be taken when developing DC-based therapies to induce tolerance in the context of autoimmune disease.

Ly49E expression on CD8αα-expressing intestinal intraepithelial lymphocytes plays no detectable role in the development and progression of experimentally induced inflammatory bowel diseases.

The Ly49E NK receptor is a unique inhibitory receptor, presenting with a high degree of conservation among mouse strains and expression on both NK cells and intraepithelial-localised T cells. Amongst intraepithelial-localised T cells, the Ly49E receptor is abundantly expressed on CD8αα-expressing innate-like intestinal intraepithelial lymphocytes (iIELs), which contribute to front-line defense at the mucosal barrier. Inflammatory bowel diseases (IBDs), encompassing Crohn's disease and ulcerative colitis, have previously been suggested to have an autoreactive origin and to evolve from a dysbalance between regulatory and effector functions in the intestinal immune system. Here, we made use of Ly49E-deficient mice to characterize the role of Ly49E receptor expression on CD8αα-expressing iIELs in the development and progression of IBD. For this purpose we used the dextran sodium sulphate (DSS)- and trinitrobenzenesulfonic-acid (TNBS)-induced colitis models, and the TNFΔARE ileitis model. We show that Ly49E is expressed on a high proportion of CD8αα-positive iIELs, with higher expression in the colon as compared to the small intestine. However, Ly49E expression on small intestinal and colonic iIELs does not influence the development or progression of inflammatory bowel diseases.

Contribution of the Ly49E natural killer receptor in the immune response to Plasmodium berghei infection and control of hepatic parasite development.

Natural killer (NK) cells have different roles in the host response against Plasmodium-induced malaria depending on the stage of infection. Liver NK cells have a protective role during the initial hepatic stage of infection by production of the TH1-type cytokines IFN-γ and TNF-α. In the subsequent erythrocytic stage of infection, NK cells also induce protection through Th1-type cytokines but, in addition, may also promote development of cerebral malaria via CXCR3-induction on CD8(+) T cells resulting in migration of these cells to the brain. We have recently shown that the regulatory Ly49E NK receptor is expressed on liver NK cells in particular. The main objective of this study was therefore to examine the role of Ly49E expression in the immune response upon Plasmodium berghei ANKA infection, for which we compared wild type (WT) to Ly49E knockout (KO) mice. We show that the parasitemia was higher at the early stage, i.e. at days 6-7 of Plasmodium berghei ANKA infection in Ly49E KO mice, which correlated with lower induction of CD69, IFN-γ and TNF-α in DX5(-) liver NK cells at day 5 post-infection. At later stages, these differences faded. There was also no difference in the kinetics and the percentage of cerebral malaria development and in lymphocyte CXCR3 expression in WT versus Ly49E KO mice. Collectively, we show that the immune response against Plasmodium berghei ANKA infection is not drastically affected in Ly49E KO mice. Although NK cells play a crucial role in Plasmodium infection and Ly49E is highly expressed on liver NK cells, the Ly49E NK receptor only has a temporarily role in the immune control of this parasite.

Abundant stage-dependent Ly49E expression by liver NK cells is not essential for their differentiation and function.

The NKR Ly49E has several unique characteristics. Unlike most NKRs, Ly49E is highly expressed on fetal NK cells, whereas expression is decreased on bone marrow-derived NK cells in adult mice. To investigate a possible role for Ly49E in NK cell differentiation and function, we have generated an Ly49E KO mouse. Our results show that bone marrow and splenic NK cells are present in normal numbers in Ly49E KO mice, expressing an unaltered panel of NKRs and differentiation markers. Furthermore, cytokine production and cytotoxicity by these cells are unaffected. Surprisingly, WT DX5(-) liver NK cells express high Ly49E levels in fetal and adult mice. Ly49E(+)DX5(-) liver NK cells transferred into Rag-2(-/-)/gc(-/-) mice maintain high Ly49E expression in the liver and differentiate into DX5(+) NK cells in spleen and bone marrow. Ly49E expression is not crucial for liver NK cell differentiation during ontogeny, as the DX5(-)/DX5(+) ratio, the NKR repertoire, and the granzyme B and TRAIL levels are comparable in Ly49E KO versus WT mice, except for lower TRAIL expression on DX5(-) liver NK cells in 20-day-old mice. The TRAIL-, perforin-, and FasL-mediated cytolysis by liver NK cells is unaffected in Ly49E KO mice. Collectively, we show that in addition to high Ly49E expression on fetal NK cells versus low Ly49E expression on conventional NK cells in adult life, Ly49E remains highly expressed on DX5(-) liver NK cells. However, Ly49E expression does not have a crucial role in differentiation and/or function of these NK cells.