Annexin-A1 Tripeptide Attenuates Surgery-Induced Neuroinflammation and Memory Deficits Through Regulation the NLRP3 Inflammasome

Abstract

Neuroinflammation is a growing hallmark of perioperative neurocognitive disorders (PNDs), including delirium and longer-lasting cognitive deficits. We have developed a clinically relevant orthopedic mouse model to study the impact of a common surgical procedure on the vulnerable brain. The mechanism underlying PNDs remains unknown. Here we evaluated the impact of surgical trauma on the NLRP3 inflammasome signaling, including the expression of apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, and IL-1β in the hippocampus of C57BL6/J male mice, adult (3-months) and aged (>18-months). Surgery triggered ASC specks formation in CA1 hippocampal microglia, but without inducing significant morphological changes in NLRP3 and ASC knockout mice. Since no therapies are currently available to treat PNDs, we assessed the neuroprotective effects of a biomimetic peptide derived from the endogenous inflammation-ending molecule, Annexin-A1 (ANXA1). We found that this peptide (ANXA1sp) inhibited postoperative NLRP3 inflammasome activation and prevented microglial activation in the hippocampus, reducing PND-like memory deficits. Together our results reveal a previously under-recognized role of hippocampal ANXA1 and NLRP3 inflammasome dysregulation in triggering postoperative neuroinflammation, offering a new target for advancing treatment of PNDs through the resolution of inflammation.

Keywords: NLRP3 inflammasome; aging; annexin A1 derived peptide; microglia; postoperative cognition dysfunction.

Figure 1

Decreased ANXA1 expression and induction of the NLRP3 inflammasome in adult mice after orthopedic surgery. Surgery reduced the expression of endogenous ANXA1 in hippocampal homogenate in a time-dependent manner, with the lowest expression found at 24-h (A). Hence, we treated mice with ANXA1sp (1 mg/kg, i.p.) 30 min before surgery and evaluated NLPR3 inflammasome activation at this timepoint. Postoperative NLRP3 inflammasome activation was significantly attenuated by ANXA1sp (B, C), as evidenced by Western blot analysis of ASC (D), cleaved caspase-1 (E), and cleaved IL-1 (F) in hippocampal homogenate. Surgery-induced hippocampal ATP was also reduced by ANXA1sp treatment (G). Data are presented as mean ± SEM. n = 4/group. *P < 0.05, **P < 0.01, ***P < 0.001; analyzed by one-way ANOVA with Bonferroni test. Representative data are from two independent experiments.

Figure 2

Effects of ANXA1sp on microglial morphology after surgery. Microglial morphologic changes were significantly curtailed 24 h after orthopedic surgery in adult mice pretreated with ANXA1sp (A, B). Microglial morphology was quantified based on four morphologic subtypes: Round, Stout, Thick, and Thin; three representative areas/mice were used for quantification and one single microglia was represented in the reconstruction (red indicates filament tracing). Data are presented as mean ± SEM from hippocampal CA1 area. n = 5/group. *P < 0.05, ***P < 0.001. Analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Scale bar: 20 μm. Representative data are from two independent experiments.

Figure 3

Postoperative neuroinflammation in Nlrp3^-/- mice. The NLRP3 inflammasome was not activated in the hippocampus of these animals at 24 h post orthopedic surgery (A, B). Moreover, no changes in microglial morphology were observed in Nlrp3^-/- mice after orthopedic surgery, as evidenced by IBA-1 immunostaining (C, D). Microglial representative filaments are provided from age matched C57BL6/J mice (WT) as comparison. For quantitation of WT data, refer to Figure 2B. Three representative areas/mice were used for quantification and one single microglia was represented in the reconstruction (red indicates filament tracing) from hippocampal CA1 area of Nlrp3^-/- mice only. Data are presented as mean ± SEM. n = 5-6/group. Scale bar: 20 μm. Representative data are from one experiment.

Figure 4

ASC speck formation after surgery. Asc^-/- showed no evidence of NLRP3 inflammasome activation in hippocampal lysate (A–C). ASC-cit reporter mice showed significant speck formation in the hippocampus, including co-expression with IBA-1-positive cells (D–F). Three representative areas/mice were used for quantification and one single microglia was represented in the reconstruction (red indicates filament tracing) from hippocampal CA1 area. Age matched WT controls are described in Figures 1 and 2. Data are presented as mean ± SEM. n = 3-5/group. **P < 0.01, ***P < 0.001; analyzed by two-tailed unpaired Student’s t test. Scale bar: 20 μm. Representative data are from one experiment.

Figure 5

PND-like cognitive responses in adult mice treated prior to surgery with the vehicle or ANXA1sp. Example of Set A and Set B objects used in the “What-Where-When” memory task (A). Example of the seven unique objects used in the Memory Load task (B). Preference scores for “What” object recognition memory in the “What-Where-When” task (C). Preference scores for “Where” recognition memory for the displaced object in the “What-Where-When” task (D). Preference scores for “When” recognition memory for recent objects to previously investigated objects in the “What-Where-When” task (E). The time (s) spent exploring Set A and Set B objects, and at testing in the “What-Where-When” task (F). Preference scores for the novel object in the Memory Load test (G). Mean time (adjusted by trial length) that animals explored objects in the seven consecutive trials in the Memory Load test (H). N = 10 mice/treatment group for all tests. *P < 0.05, compared to vehicle + surgery mice; ⁺P < 0.05, compared to naïve animals. Results are shown as mean ± SEM and analyzed by MANOVA. Representative data are from one experiment.

Figure 6

Effects of ANXA1sp on NLRP3 inflammasome and microglial morphology in aged mice after surgery. The hippocampal NLRP3 inflammasome complex was significantly activated in a time-dependent manner in aged mice after orthopedic surgery, with older mice showing a significantly higher baseline compared to adult mice (A). Hippocampal NLRP3 inflammasome activation (B–F) was attenuated in aged mice treated with ANXA1sp (1 mg/kg, ip) 30 min before orthopedic surgery (same dose and timepoint as in the adult mice). Similarly, microglial activation in the CA1 area was reduced 24 h after surgery (G, H), especially improving ameboidal morphologies (round, stout, and think). Three representative areas/mice were used for quantification and one single microglia was represented in the reconstruction (red indicates filament tracing). Hippocampal ATP expression (I) was also restored in aged mice treated with ANXA1sp. Data are presented as mean ± SEM. n = 5. **P < 0.01, ***P < 0.001, compared to naïve or vehicle controls. Analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Scale bar: 20 μm. Representative data are from two independent experiments. *P < 0.05.

Figure 7

Proposed NLPR3 signaling mechanism in postoperative neuroinflammation. Aseptic surgical trauma induces a cytokine storm that engages several pattern recognition receptors (PRRs), including the interlukin-1 (IL-1) receptor. Downstream signaling from these receptors relies on adaptor proteins such as MyD88, which eventually activates NF-κB to perpetuate the inflammatory response. Parallel to this process, surgery also elevated levels of ATP in the hippocampus. In combination with NF-κB activation this induced the NLRP3 inflammasome and ASC specks formation in microglia, thus contributing to postoperative neuroinflammation. Surgery also impaired the expression of hippocampal Annexin-A1 (ANXA1) that is critically involved in inflammation-resolution and can be regulated through our biomimetic peptide. Within this framework we found that treatment with ANXA1sp regulated NLRP3 expression and levels of IL-1β postoperatively. Given the established elevation of IL-1β in preclinical and clinical biofluids of subjects with postoperative neurocognitive disorders, harnessing this pathway may provide safer strategies to treat ensuing neuroinflammation.