Interleukin-8 (IL-8) is a key chemokine upregulated in various forms of intestinal inflammation, especially those induced by bacteria such as Clostridium difficile (C. difficile). Although interactions between different mucosal and submucosal cellular components have been reported, whether such interactions are involved in the regulation of IL-8 secretion during C. difficile infection is unknown. Moreover, whether the enteric nervous system, a major component of the submucosa, is involved in IL-8 secretion during an inflammatory challenge remains to be determined. In order to investigate mucosa/submucosa interactions that regulate IL-8 secretion, we co-cultured human intestinal mucosa and submucosa. In control condition, IL-8 secretion in co-culture was lower than the sum of the IL-8 secretion of both tissue layers cultured alone. Contrastingly, IL-8 secretion increased in co-culture after mucosal challenge with toxin B of C. difficile through an IL-1 beta-dependent pathway. Moreover, we observed that toxin B of C. difficile increased IL-8 immunoreactivity in submucosal enteric neurones in co-culture and in intact preparations of mucosa/submucosa, through an IL-1 beta-dependent pathway. IL-1 beta also increased IL-8 secretion and IL-8 mRNA expression in human neuronal cell lines (NT2-N and SH-SY5Y), through p38 and ERK1/2 MAP kinase-dependent pathways. Our results demonstrate that mucosa/submucosa interactions regulate IL-8 secretion during inflammatory processes in human through IL-1 beta-dependent pathways. Finally we observed that human submucosal neurones synthesize IL-8, whose production in neurones is induced by IL-1 beta via MAPK-dependent pathways.