Hepatic stellate cells (HSCs) have potent immunoregulatory properties and have been shown to induce myeloid cells with suppressive function, which contribute to liver transplantation tolerance. HSCs store retinol and are an important source of retinoic acid (RA). RA is known to be a regulator of the immune response and to endow dendritic cells (DCs) with T cell immunosuppressive function. Hence, we hypothesized that HSC-derived RA may be partially responsible for the development of DCs with T cell suppressive function. RA treatment of bone marrow cells cultured with GM-CSF and IL-4 (RA-DCs) resulted in the generation of a cell population with phenotype and morphology similar to DCs (CD11c+CD11b+MHC IIhi). However, RA-DCs potently suppressed DC-stimulated OT-II T cell proliferation. Compared to DCs, RA-DCs exhibited increased expression of arginase 1 (Arg1) but not inducible nitric oxide synthase (iNOS). Administration of the arginase inhibitor (nor-NOHA) and/or iNOS inhibitor (L-NMMA) each partially reversed RA-DC-mediated suppression of T cell proliferation. To test the contribution of Arg1 and iNOS further, RA-DCs were generated from iNOS knockout mice and co-cultured with DC-stimulated OT-II T cells. Interestingly, suppressive function was significantly reduced in iNOS-/- RA-DCs. Expression of iNOS in RA-DCs and T cell suppression by RA-DCs required IFN-Γ sensitivity since IFN-Γ receptor deficient RA-DCs produced lower levels of NO and had reduced suppressive function compared to wildtype RA-DCs. Furthermore, iNOS mRNA expression was increased in RA-DCs compared to DCs following stimulation with IFN-Γ. These observations suggest that RA can directly induce Arg1 and sensitize DCs for IFN-Γ-induced iNOS production. We propose that T cell-derived IFN-Γ can induce iNOS in RA-DCs, which along with Arg1, can suppress T cell proliferation.

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