*Purpose: Liver conventional myeloid (m)DC contribute to liver transplant tolerance. When freshly-isolated, they produce low levels of IL-12 but high levels of IL-10 compared with lymphoid tissue mDC. Exposure to IL-10, that is produced constitutively by various liver cell populations, potentiates their T cell tolerogenicity. Our previous data have linked expression of the transmembrane adaptor protein DNAX-activating protein of 12KDa (DAP12, a key regulator of liver mDC function, with that of IL-1R-associated kinase (IRAK)-M, a negative regulator of TLR signaling. We postulated that exposure to IL-10 would promote acquisition of an LPS-refractory/tolerogenic state in liver mDC via DAP12-regulated IRAK-M and IL-10 expression.

*Methods: Wild-type (WT) and DAP12^-/- B6 mouse liver mDC (CD11b+NK1.1-CD11c+) were isolated and exposed to LPS in the absence or presence of rm IL-10+/- IL-10R neutralizing antibody. Phenotypic analysis was performed by flow cytometry to characterize the expression of MHC II, CD80, CD86 and PD-L1. IRAK-M and nuclear factor (NF) kB/p-NFkB expression were probed by Western blot. mRNA transcripts and cytokine levels in supernatants were quantified using q-PCR and mouse inflammation CBA kits respectively. T cell allostimulatory function of the liver mDC was assessed in CFSE-MLR.

*Results: IL-10 exposure increased expression of IRAK-M and diminished that of NFkB/p-NFkB by WT liver mDC, reduced levels of MHC II and co-stimulatory molecule expression, but increased levels of co-inhibitory PD-L1, with/without LPS stimulation. In addition, TNF-α and IL-12 secretion was attenuated, whereas IL-10 secretion was enhanced. IL-10-treated liver mDC also showed inferior ability to stimulate allogeneic T cell proliferation. Compared with WT liver mDC, DAP12^-/- liver mDC showed lower expression of IRAK-M with/without LPS stimulation, , higher levels of CD80 and CD86 but reduced PD-L1 expression and augmented TNF-α/IL-12 secretion. Unlike with WT liver mDC, IL-10 exposure did not significantly affect IRAK-M expression, co-stimulatory molecules or pro-inflammatory cytokine production by DAP12^-/- liver mDC.

*Conclusions: DAP12-regulated IRAK-M expression and enhanced IL-10 production underlie the IL-10-induced LPS-refractory/tolerogenic state of liver mDC.

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