Since brain cytokine expression was comparable between FK565 and MDP, it appears unlikely that the FK565-evoked rise of plasma corticosterone was mediated by cytokines. Since nitric oxide (NO) participates in the activation of the HPA axis (Bugajski et al., 2004) and FK565 is more potent in inducing NO than MDP (Cartwright et al., 2007), NO may be a mediator of the cytokine-independent HPA axis stimulation due to NOD1 agonism. As MDP and FK565 were also unable to change body temperature, anxiety-like behavior and SP, we conclude that stimulation of NOD1 and NOD2 alone,

with doses of FK565 and MDP that enhance the effects of LPS, is insufficient to evoke an overt sickness response. Interaction and crosstalk between the signaling pathways of TLRs and NLRs lead to increased or decreased production HCS assay of proinflammatory cytokines, depending on the cell type tested (Elinav et al., 2011). Pretreatment of monocytic cells with NOD agonists can facilitate the LPS-induced production of various cytokines (Chamaillard et al., 2003, Fritz et

al., 2005, Park et al., 2007 and Uehara et al., 2005), and a similar synergistic increase of cytokine production following exposure to NLR and TLR agonists is seen in vivo ( Parant et al., 1995 and Shikama et al., 2011). Furthermore, priming with MDP enhances anaphylactoid reactions and lethality evoked by LPS ( Takada and Galanos, 1987 and Takada et al., 1990), while intravenous administration Kinase Inhibitor Library mouse of FK565 alone has been reported to elicit signs of septic shock in rats ( Cartwright et al., 2007). Priming with MDP can also aggravate the reduction of ingestion and locomotion induced by LPS in rats ( Engeland et al., 2003 and Langhans et al., 1990), whereas the behavioral effects of combined NOD1 and TLR4 agonism remained unexplored. The ability of NLR agonism to aggravate and prolong the sickness response to LPS is particularly highlighted by the LabMaster data. Specifically, the low dose of 0.1 mg/kg LPS was able to decrease only

locomotion and ingestion, while the combination of FK565 + LPS and MDP + LPS aggravated and prolonged the effects of TNF-alpha inhibitor LPS on all parameters tested (locomotion, exploration, ingestion, SP) and led to a significant decrease of locomotion, exploration (rearing) and food intake for 2–3 days. In contrast, SP was decreased for a shorter period of time. The LabMaster results also shed some light on the effect of single housing in immune–brain interactions. Housing conditions can modify affective behavior (Painsipp et al., 2011), and single housing made the animals more vulnerable by the PRR agonists. While, in line with the literature (Frenois et al., 2007), novelty-induced locomotion in the OF was not altered 1 day after treatment with 0.1 mg/kg LPS, home cage activity in the LabMaster was decreased for a longer period. Since avoidance of physical activity is a sensitive indicator of illness (Skinner et al.