AbstractBackground & AimsInflammation promotes the progression of non‐alcoholic steatohepatitis (NASH). Toll‐like receptor 4 (TLR4) and TLR9 activation through myeloid differentiation primary response gene 88 (MyD88) and production of mature interleukin‐1β (IL‐1β) via inflammasome activation contribute to steatohepatitis. Here, we investigated the inter‐relationship between TLR signalling and inflammasome activation in dietary steatohepatitis.MethodsWild type (WT), TLR4‐ and MyD88‐deficient (KO) mice received methionine‐choline‐deficient (MCD) or ‐supplemented (MCS) diets for 5 weeks and a subset was challenged with TLR9 ligand CpG‐DNA.ResultsTLR4, TLR9, AIM2 (absent in melanoma 2) and NLRP3 (NLR family pyrin domain containing 3) inflammasome mRNA, and mature IL‐1β protein levels were increased in MCD diet‐induced steatohepatitis compared to MCS controls. TLR9 stimulation resulted in greater up‐regulation of the DNA‐sensing AIM2 expression and IL‐1β production in livers of MCD compared to MCS diet‐fed mice. High mobility group box 1 (HMGB1), a TLR9‐activating danger molecule and phospho‐HMGB1 protein levels were also increased in livers of MCD diet‐fed mice. MyD88‐ but not TLR4‐deficiency prevented up‐regulation of AIM2, NLRP3 mRNA and IL‐1β protein production in dietary steatohepatitis. Selective MyD88 deficiency either in bone marrow (BM)‐derived or non‐BM‐derived cells attenuated hepatic up‐regulation of inflammasome mRNA, caspase‐1 activation and IL‐1β protein production, but only BM‐derived cell‐specific MyD88‐deficiency attenuated liver injury.ConclusionsOur data demonstrate that both bone marrow‐derived and non‐BM‐derived cells contribute to inflammasome activation in a MyD88‐dependent manner in dietary steatohepatitis. We show that AIM2 inflammasome expression and activation are further augmented by TLR9 ligands in dietary steatohepatitis.