Blockade of IL-17A/IL-17R Pathway Protected Mice from Sepsis-Associated Encephalopathy by Inhibition of Microglia Activation

Abstract

Sepsis-associated encephalopathy (SAE) is a poorly understood condition that leads to long-term cognitive impairment and increased mortality in survivors. Recent research revealed that IL-17A/IL-17R might serve as a checkpoint in microglia-mediated neuroinflammation. The present study was designed to determine the specific role of IL-17A-mediated microglia activation in the development of SAE. A mouse model of SAE was induced by cecal ligation and puncture (CLP), and behavior performance was evaluated by the inhibitory avoidance test and the open field test. Cytokine expression and microglia activation in brain tissue were determined at 6 h, 12 h, 24 h, 48 h, and day 7 post surgery. Further, septic mice were intracerebral ventricle- (i.c.v.-) injected with recombinant IL-17A, anti-IL-17A ab, anti-IL-17R ab, or isotype controls to evaluate the potential effects of IL-17A/IL-17R blockade in the prevention of SAE. Septic peritonitis induced significant impairment of learning memory and exploratory activity, which was associated with a higher expression of IL-17A, IL-1β, and TNF-α in the brain homogenate. Fluorescence intensity of Iba-1 and IL-17R in the hippocampus was significantly increased following CLP. Treatment with recombinant IL-17A enhanced the neuroinflammation and microglia activation in CLP mice. On the contrary, neutralizing anti-IL-17A or anti-IL-17R antibodies mitigated the CNS inflammation and microglia activation, thus alleviating the cognitive dysfunction. Furthermore, as compared to the sham control, microglia cultured from CLP mice produced significantly higher levels of cytokines and expressed with higher fluorescence intensity of Iba-1 in response to IL-17A or LPS. Pretreatment with anti-IL-17R ab suppressed the Iba-1 expression and cytokine production in microglia stimulated by IL-17A. In conclusion, blockade of the IL-17A/IL-17R pathway inhibited microglia activation and neuroinflammation, thereby partially reversing sepsis-induced cognitive impairment. The present study suggested that the IL-17A/IL-17R signaling pathway had an important, nonredundant role in the development of SAE.

Figure 1

(a) Behavior performance was evaluated by the inhibitory avoidance test and the open field test at various time points following sham or CLP procedures. Each experiment was performed in triplicate and repeated at least twice (n = 5‐7 per group). (b) Cytokines of IL-17A, IL-1β, and TNF-α in brain homogenate were determined by ELISA. ^∗P < 0.05vs. sham control, ^#P < 0.05 CLP day 7 vs. CLP 48 h, n = 5‐6 per group.

Figure 2

Microglia activation in the hippocampus region was determined by IF at 6 h, 12 h, 24 h, 48 h, and day 7 post surgery. (a) Representative image of the IF assay in the hippocampus. Specimens were stained with CD11b (green), Iba-1 (red), and DAPI (blue). Representative images were merged and analyzed (1 : 200). Mean fluorescence intensity (MFI) of Iba-1 was shown in each image. (b) Summary data of CD11b and Iba-1 expression in the hippocampus region (n = 5‐6). ^∗P < 0.05vs. sham control; ^#P < 0.05 CLP day 7 vs. CLP 48 h.

Figure 3

IL-17R expression in the hippocampus region was determined by IF at 6 h, 12 h, 24 h, 48 h, and day 7 post surgery. (a) Representative image of the IF assay in the hippocampus. Specimens were stained with IL-17R (green), Iba-1 (red), and DAPI (blue). Representative images were merged and analyzed (1 : 200). Mean fluorescence intensity (MFI) of IL-17R was shown in each image. (b) Summary data of IL-17R and Iba-1 expression in the hippocampus region (n = 5‐6). ^∗P < 0.05vs. sham control; ^#P < 0.05 CLP day 7 vs. CLP 48 h.

Figure 4

Behavior performance and neuroinflammation were determined following the modulation of the IL-17A/IL-17R pathway. Mice received i.c.v. injection of recombinant IL-17A (20 ng), anti-IL17A ab (2 μg), anti-IL-17R ab (1 μg), isotype control (isotypeA: IgG1Ƙ 2 μg, isotypeB: IgG2A 1 μg), or saline. Samples were collected and analyzed at 12 h, 48 h, or 7 days post surgery. ^∗P < 0.05vs. sham control; ^#P < 0.05 (CLP+anti-IL17A ab vs. CLP+isotypeA; CLP+anti-IL17R ab vs. CLP+isotypeB).

Figure 5

Microglia activation was determined by IF after modulation of the IL-17A/IL-17R pathway. (a) Representative image of the IF assay in the hippocampus at 12 hours post surgery (1 : 200). Specimens were stained with CD11b (green), Iba-1 (red), and DAPI (blue). Mean fluorescence intensity (MFI) of Iba-1 was shown in each image. (b) Summary data of the IF assay at 12 hours, 48 hours, and day 7 post CLP. ^∗P < 0.05vs. sham control; ^#P < 0.05 (CLP+anti-IL17A ab vs. CLP+isotypeA; CLP+anti-IL17R ab vs. CLP+isotypeB).

Figure 6

(a) Primary microglial cells were collected and cultured with the purity of over 90%. Cells were stained with anti-CD11b antibody and examined by flow cytometry. (b) IL-17A or LPS induced cytokine production in microglia in vitro. Microglia cultured from CLP or sham-operated mice was pretreated with anti-IL-17R ab or isotype control and then stimulated with or without IL-17A (50 ng/ml or 100 ng/ml) or LPS (1 μg/ml). Supernatant was collected 24 h after stimulation and measured by ELISA. (c) Mean fluorescence intensity of Iba-1 in cultured microglia was determined by flow cytometry. ^∗P < 0.05 vs. PBS control (sham group); ^#P < 0.05 vs. PBS control (CLP group); ^†P < 0.05 CLP vs. sham; ^$P < 0.05 vs. IL-17A 100 ng/ml (CLP group); ^‡P < 0.05 vs. IL-17A 100 ng/ml (sham group).