This study found that peripheral surgery induced an increase of IL-1β in the plasma and hippocampus, which also activated monocytes (Ly6Chigh) in peripheral blood and increases intracellular levels of IL-1β. We also found that when NLRP3 expression was inhibited by AAV, increased IL-1R1, IL-1β, IL-18 expression, and microglia activation were all downregulated in the hippocampus. Therefore, we deduced that there was an IL-1R1/NLRP3/IL-1β inflammatory feedback mechanism in the hippocampus, inhibiting the feedback can reduce the peripheral surgery-induced hippocampus inflammatory response and brain injury.
We performed a carotid artery exploration surgery to induce an inflammatory response and detect inflammatory factors after surgery(23, 24). The results showed that IL-1β expression significantly increased in the peripheral system 30 minutes after surgery, and gradually decreased after 12 hours. Tissue injury and cell death could induce activated monocytes mobilized from the bone marrow into the blood rapidly(25). We studied the effects of surgery on the monocyte count and its intracellular IL-1β expression, the result showed that the number of labeled monocytes by Ly6Chigh increased after peripheral surgery. The intracellular IL-1β remained much higher 24 hours postoperatively than the control group. Previous studies showed that Ly6Chigh monocytes migrated through the BBB into the CNS and differentiate gradually into microglia in some cases of infection and injury(7, 26). However, since the migration of peripheral monocytes to the CNS was not observed in this study, more researches would be needed to verify this. Furthermore, some studies have shown that Ly6Chigh monocytes had an extremely short half-life in the bloodstream of only 19 hours, our result showed a similar result(27). In a study of the regulation of inflammation by human monocytes, the authors claimed that monocytes were central to the regulation of inflammation in response to infection or injury, and IL-1β generated by monocytes was crucial to the initiation and maintenance of inflammatory responses(28). In conclusion, we speculate that IL-1β generated by monocytes has a continuous and persistent impact on Peripheral surgery-mediated inflammatory responses.
Activated monocytes played an important role in the inflammation caused by surgery. Monocytes generating IL-1β were recruited by the microglial cell to the brain cause stress-induced anxiety by binding of IL-1β to IL-1R1 in a stress-induced anxiety animal model(29). Our research discovered that IL-1β levels in the brain increased dramatically after surgery, accompanied by an increase in IL-1R1, and NLRP3 protein levels, as well as neuron damage detected by Nissl's staining. Since the brain was the most significant innate immune organ and contained a large number of inflammatory factor receptors, such as IL-1R1, and it was a crucial target for surgery-induced inflammatory response. Additionally, IL-1β was a pivotal proinflammatory cytokine and belonged to the interleukin-1 family, consisting of 11 unique known ligands and antagonistic receptors(30). It could activate immune cells in CNS such as microglia, and promote neuronal cell damage to impair brain function via glutamate production(12, 31). We observed that IL-1R1 expression in the hippocampus started at 6 hours after surgery, NLRP3 rise at 12 hours, and IL-1β peaked at 18 hours after surgery. Meanwhile, fluorescence microscopy experiments showed that NLRP3 formed multiple small puncta upon surgery treatment after 18 hours, and this implies that NLRP3 has been activated. Chen et al. reported that NLRP3 primarily aggregates into several small puncta before being combined into a big speck with ASC, and these puncta were verified to be the active form of NLRP3 in their cell experiments(32). Recent studies suggested that IL-1β was not only a downstream inflammatory factor produced by the NLRP3 inflammasome but also an inflammatory signal that promotes the synthesis of NLRP3 in the nucleus via the IL-1R1(15, 33, 34). We assume that there is a feedback mechanism about IL-1β-IL-1R1-NLRP3 inflammasome existing in the pathological process of brain injury induced by peripheral surgery.
Some studies demonstrated that the activation of NLRP3 inflammasome generates a large amount of IL-1β, which was required for neuroinflammation in the development of neuron damage(35–37). In a study of Biliary Atresia (BA), the authors blocked the IL-1R1-NLRP3-IL-1 signaling pathway by inhibiting NLRP3, which decreased immune cell activation and the progression of BA(38). We inhibited the expression of NLRP3 by injecting AAV into the encephalocele. The results demonstrated a substantial decrease in inflammatory markers, IL-1R1, and NLRP3 protein in the brain, a decrease in cell damage, and downregulated microglia's expression. Based on this, we assumed that IL-1β, induced by surgical trauma, could bond with IL-1R1 in the CNS and lead to generate more inflammatory factors via activating NLRP3 inflammasome, which in turn causes microglia to initiate the inflammatory cascade and damage neurons. Inhibiting NLRP3 expression could block the feedback mechanism about IL-1β-IL-1R1-NLRP3.
However, we were not in a position to determine how IL-1β enters the CNS: Either the monocytes were released directly into the blood vessels or the monocytes first passed through the BBB and then released IL-1β. Therefore, the definite effect of monocyte action needs to be further studied. One limitation of this research is that we have focused primarily on the effect of IL-1β on NLRP3 while ignoring other inflammatory factors, such as TNF-α also induces activation of NLRP3 inflammasome. In another word, the entire inflammatory cascade was a huge and complicated system consisting of complicated inflammatory cytokines and immune cells. Therefore, the process has yet to be clearly and detailedly explored. In addition, although NLRP3-induced neuroinflammation could be effectively alleviated with AAV-NLRP3, there were still too many other conditions to be a clinical drug suitable for widespread application. Future research would focus on identifying a suitable drug to replace AAV-NLRP3 to prevent post-operative neuroinflammation. At the same time, our experiment used young mice to determine if this inflammatory route existed. In the future, we would further study the relationship between this inflammatory pathway and POCD in aged mice, and whether blocking this pathway can affect behavioral changes in aged mice.
In conclusion, the peripheral surgery induced an increase of IL-1β, IL-1R1, NLRP3, and neuron injury, inhibiting the expression of NLRP3 could alleviate inflammatory factors expression and cell damage. We assumed that there was a feedback mechanism about IL-1β-IL-1R1-NLRP3 inflammasome existing in the pathological process of brain injury induced by peripheral surgery.