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Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system.

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Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, has been shown to induce profound changes both peripherally and centrally. It has recently been reported that intraperitoneal injection of LPS inhibited long term potentiation (LTP) in perforant path-granule cell synapses and that this effect was coupled with an increase in the concentration of the proinflammatory cytokine, interleukin-1␤ (IL-1␤). The LPS-induced effects were abrogated by inhibition of caspase-1, suggesting that IL-1␤ may mediate the effects of LPS. Here we report that the inhibition of LTP induced by LPS and IL-1␤ was coupled with stimulation of the stress-activated protein kinase p38 in hippocampus and entorhinal cortex and that this effect was abrogated by the p38 inhibitor SB203580, while the effect of LPS was markedly attenuated in C57BL/6 IL-1RI ؊/؊ mice. The data also indicate that activation of the transcription factor, nuclear factor B (NFB), may play a role, since the inhibitory effect of LPS and IL-1␤ on LTP was attenuated by the NFB inhibitor, SN50; consistently, LPS and IL-1␤ led to activation of NFB in entorhinal cortex. We suggest that one consequence of these LPSand IL-1␤-induced changes is a compromise in glutamate release in dentate gyrus, which was coupled with the inhibition of LTP. The evidence is consistent with the idea that the LPS-induced impairment in LTP is mediated by IL-1␤ and is a consequence of activation of p38.The proinflammatory cytokine, interleukin-1␤ (IL-1␤), 1 exerts numerous effects in the central nervous system; among these effects is inhibition of long term potentiation (LTP) in the hippocampus (1-6). LTP in perforant path-granule cell synapses has been shown to be attenuated in aged and stressed rats (4, 5) and in rats that were treated intraperitoneally with lipopolysaccharide (LPS; Ref. 7) or intracerebroventricularly with IL-1␤ (4, 5); IL-1␤ concentration in hippocampus was increased in each of these experimental conditions, and a negative correlation between the ability of rats to sustain LTP and IL-1␤ concentration in hippocampus has been described previously (8, 9). The finding that the effect of LPS on LTP is mimicked by IL-1␤, together with the finding that the effect of LPS on LTP was abrogated by inhibiting IL-1␤ converting enzyme (caspase-1; Ref. 7), suggested that IL-1␤ may mediate this effect of LPS.Among the downstream effects of IL-1␤ is activation of the mitogen-activated protein kinases, c-Jun NH 2 -terminal kinase (JNK), or stress-activated protein kinase (SAPK) and p38; this effect has been reported in several tissues, for example, IL-1␤ increases activity of p38 in Chinese hamster CCl39 (10) and HeLa (11) cells, while IL-1␤-induced activation of JNK has been reported in human glomerular mesangial (12) and HeLa (11) cells. In the hippocampus, activation of JNK and p38 is coupled with elevated IL-1␤ concentrations in aged rats (13) and in rats treated intracerebroventricularly with IL-1␤ (6), and the LTP-associated increase in KCl-stimulated glutamate rele...