Caspase-1 Activation Contributes To Nod2-mideated Tolerance In Microglia

Background Microglia, the crucial immunological cell in the central nervous system (CNS), has been recognized as potentially important contributor to inflammatory immune responses within the brain. Exposure of microglia to inflammatory molecules, microbial challenge and bacterial components, has been demonstrated to induce the production of inflammatory cytokines. Numerous demonstrations have revealed that microglia expressed members of Toll-like family of pattern recognition receptors (TLRs) and nucleotide-binding domain leucine-rich repeat region-containing family of proteins (NLRs). NOD2 was one vital member of NLRs that played a pivotal role in the detection of bacterial CNS pathogens and the initiation of inflammation within the brain. Demonstrations have reported that NOD2 was a general sensor of bacterial peptidoglycans as it recognized a minimal motif, Muramyl dipeptide (MDP), presenting in all peptidoglycans. Activation upon Nod2 stimulation induced inflammatory cytokine production, such as TNF-α and IL-6, through the NF-κB pathway.Although the production of inflammatory cytokines served as an important mechanism in protecting the host from bacterial, excessive production of these cytokines without proper regulation is detrimental to the host and may lead to microcirculatory dysfunction and tissue damage. NOD2-mediated tolerance in macrophage was one protective mechanism during bacterial infection. It was unclear, however, whether NOD2-mediated tolerance worked in microglia.Nod2 has also been implicated in regulating the activation of Caspase-1.Caspase-1 was a key mediator of host immune responses and played an important role in adaptive and innate immunity. In addition to the inflammatory challenge and bacterial components, caspase-1 could be activated by ATP to induce the cytokine production for the adjustment of the immune response which protecting organism from bacterial infection or injury. Therefore, it is significant to investigate whether the NOD2-mediated tolerance phenomenon exists in microglia cells and its relationship with Caspase-1.Objective To investigate whether the NOD2- mediated tolerance phenomenon exists in microglia cells and its relationship with Caspase-1.Methods After microglia cells were stimulated with different concentrations of MDP or S.pneumonia for 24h, the expressions of NOD2 and NF-κB were detected by Western blotting and the expressions of IL-6 and TNF-α were detected by ELISA. After microglia cells were pre-treated with MDP (100μg/mL) for 0,3 or 24h, the expressions of IL-6 and TNF-a were detected by ELISA to verify the NOD2 mediated tolerance in microglia cells. After Caspase-1 activity was activated or suppressed by ATP or Caspase-1 inhibitor, the expressions of IL-6 and TNF-a were detected by ELISA to observe the relationship between NOD2 mediated tolerance and Caspase-1.Results Compared with no treatment, MDP and S.pneumonia treatment for 24h significantly increased the levels of NOD2, NF-κB, IL-6 and TNF-a in microglia cells (P<0.05). MDP pretreatment for 3 or 24h reduced secretion of IL-6 and TNF-a upon subsequent stimulation of MDP for 24h. Levels of IL-6 and TNF-a were significantly down regulated by ATP pretreatment, while up-regulated by Caspase-1 inhibitor treatment.Conclusion Prolonged periods of MDP exposure can induce NOD2- mediated tolerance in microglia cells and NOD2- mediated tolerance is associated with Caspase-1 activity.