A systematic analysis of the peripheral and CNS effects of systemic LPS, IL-1β, [corrected] TNF-α and IL-6 challenges in C57BL/6 mice

Abstract

It is increasingly clear that systemic inflammation has both adaptive and deleterious effects on the brain. However, detailed comparisons of brain effects of systemic challenges with different pro-inflammatory cytokines are lacking. In the present study, we challenged female C57BL/6 mice intraperitoneally with LPS (100 µg/kg), IL-1β (15 or 50 µg/kg), TNF-α (50 or 250 µg/kg) or IL-6 (50 or 125 µg/kg). We investigated effects on core body temperature, open field activity and plasma levels of inflammatory markers at 2 hours post injection. We also examined levels of hepatic, hypothalamic and hippocampal inflammatory cytokine transcripts. Hypothermia and locomotor hypoactivity were induced by LPS>IL-1β>TNF-α>>IL-6. Systemic LPS, IL-1β and TNF-α challenges induced robust and broadly similar systemic and central inflammation compared to IL-6, which showed limited effects, but did induce a hepatic acute phase response. Important exceptions included IFNβ, which could only be induced by LPS. Systemic IL-1β could not induce significant blood TNF-α, but induced CNS TNF-α mRNA, while systemic TNF-α could induce IL-1β in blood and brain. Differences between IL-1β and TNF-α-induced hippocampal profiles, specifically for IL-6 and CXCL1 prompted a temporal analysis of systemic and central responses at 1, 2, 4, 8 and 24 hours, which revealed that IL-1β and TNF-α both induced the chemokines CXCL1 and CCL2 but only IL-1β induced the pentraxin PTX3. Expression of COX-2, CXCL1 and CCL2, with nuclear localisation of the p65 subunit of NFκB, in the cerebrovasculature was demonstrated by immunohistochemistry. Furthermore, we used cFOS immunohistochemistry to show that LPS, IL-1β and to a lesser degree, TNF-α activated the central nucleus of the amygdala. Given the increasing attention in the clinical literautre on correlating specific systemic inflammatory mediators with neurological or neuropsychiatric conditions and complications, these data will provide a useful resource on the likely CNS inflammatory profiles resulting from systemic elevation of particular cytokines.

Figure 1. Impact of systemic LPS, IL-1β, TNF-α and IL-6 treatment on core body temperature and locomotor activity.

Deviation in core-body temperature from baseline (a) and in locomotor activity (as a percentage of baseline activity at t=0) (b) were assessed 2 hours after systemic challenge (i.p.) with saline, LPS (100 µg/kg), IL-1β (15 µg/kg or 50 µg/kg), TNF-α (50 µg/kg or 250 µg/kg) and IL-6 (50 µg/kg or 125 µg/kg). Data were analysed by one-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between saline, LPS and each cytokine treatment at the higher dose. # denotes treatment is significantly different to saline control ( # p<0.05 # # p<0.01, # # # p<0.001). * denotes differences between treatments p < 0.05 (n=5 for all groups, except LPS n=4 and 15 µg/kg IL-1β n=3). + denotes significant difference between low and high dose of cytokine p<0.05. All data have been presented as mean ± SEM.

Figure 2. Impact of systemic LPS, IL-1β, TNF-α and IL-6 challenge on plasma inflammatory and stress mediators.

IL-1β (a), TNF-α (b), IL-6 (c), IL-1ra (d), corticosterone (e) and PGEM (f) as measured by ELISA in plasma prepared from whole blood of C57BL/6 female mice 2 hours after systemic challenge (i.p.) with saline, LPS (100 µg/kg), IL-1β (15 µg/kg or 50 µg/kg), TNF-α (50 µg/kg or 250 µg/kg) or IL-6 (50 µg/kg or 125 µg/kg). All data groups were compared by one-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between saline, LPS and each cytokine treatment at the higher dose. Endogenous versus injected levels could not be discriminated for IL-1β, TNF-α or IL-6 (denoted peripherally administered). # denotes treatment is significantly different to saline control # p<0.05. ** denotes significant difference between treatment groups indicated by line p < 0.01, ***p < 0.001. All data have been presented as mean ± SEM. n=5 for all groups except LPS and IL-1β 50 µg/kg n=4 and IL-1β 15 µg/kg n=3.

Figure 3. Impact of systemic LPS, IL-1β, TNF-α and IL-6 challenge on mRNA transcription of hepatic pro-inflammatory cytokines IL-1β, TNF-α and downstream genes.

Liver transcription of mRNA species for IL-1β, TNF-α, CXCL1, CCL2, SAA2 and TNF-αIP2 were measured using quantitative PCR in C57BL/6 female mice 2 hours after systemic challenge (i.p.) with saline, LPS (100 µg/kg), IL-1β (15 µg/kg or 50 µg/kg), TNF-α (50 µg/kg or 250 µg/kg) and IL-6 (50 µg/kg or 125 µg/kg). Data were analysed by one-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between saline, LPS and each cytokine treatment at the higher dose. ##, denotes treatment is significantly different to saline treatment # # p<0.01, # # # p<0.001. * denotes significant difference between treatment groups indicated by line p < 0.05, **p<0.01, ***p<0.001. +++ denotes significant difference between low and high dose of cytokine p<0.001. All data have been presented as mean ± SEM, n=5 for all groups.

Figure 4. Impact of systemic LPS, IL-1β, TNF-α and IL-6 challenge on hypothalamic mRNA transcription.

Hypothalamic transcription of mRNA species for IL-1β, TNF-α and IL-6 assessed using quantitative PCR 2 hours after systemic challenge (i.p.) with saline, LPS (100 µg/kg), IL-1β (15 µg/kg or 50 µg/kg), TNF-α (50 µg/kg or 250 µg/kg) and IL-6 (50 µg/kg or 125 µg/kg). Data were analysed by one-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between saline, LPS and each cytokine treatment at the higher dose. ## denotes treatment is significantly different to saline treatment # # p<0.01, # # # p<0.001. ** denotes significant difference between treatment groups indicated by line p < 0.01, *** p<0.001. All data have been presented as mean ± SEM, n=5 for all groups, except IL-1β 15 µg/kg n=3.

Figure 5. Impact of systemic LPS, IL-1β, TNF-α and IL-6 challenge on hippocampal inflammatory cytokine transcription.

Hippocampal transcription of IL-1β, CXCL1, IL-6, TNF-α, IFN-α and IFN-β mRNA measured using quantitative PCR 2 hours after systemic challenge (i.p.) with saline, LPS (100 µg/kg), IL-1β (15 µg/kg or 50 µg/kg), TNF-α (50 µg/kg or 250 µg/kg) or IL-6 (50 µg/kg or 125 µg/kg). All data groups were compared by one-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between saline, LPS and each cytokine treatment at the higher dose. ## denotes treatment is significantly different to saline treatment # # p<0.01, # # # p<0.001. ** denotes significant difference between treatment groups indicated by line p < 0.01 *** p<0.001. +++ denotes significant difference between low and high dose of cytokine p<0.001. All data have been presented as mean±SEM, n=5 for LPS and IL-1β 50 µg/kg; n≥4 for TNF-α 250 µg/kg; n≥3 for other groups.

Figure 6. Impact of systemic LPS, IL-1β, TNF-α and IL-6 challenge on cyclooxygenases and cell markers.

Hippocampal transcription of the biosynthetic enzymes COX-1, COX-2, VCAM, uPAR and CD11b mRNA measured using quantitative PCR in C57BL/6 female mice 2 hours after systemic challenge (i.p.) with saline, LPS (100 µg/kg), IL-1β (15 µg/kg or 50 µg/kg), TNF-α (50 µg/kg or 250 µg/kg) and IL-6 (50 µg/kg or 125 µg/kg). All data groups were compared by one-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between saline, LPS and each cytokine treatment at the higher dose. # denotes treatment is significantly different to saline treatment p<0.05, # # p<0.01, # # # p<0.001. *** denotes significant difference between treatment groups indicated by line p < 0.001. All data have been presented as mean±SEM, n=5 for all groups except n=4 for TNF-α 250 µg/kg; n≥3 for IL-1β 15 µg/kg and both IL-6 groups.

Figure 7. Impact of systemic IL-1β or TNF-α challenge on hippocampal and hypothalamic pro-inflammatory cytokine mRNA transcription at 1, 2, 4, 8 and 24 hours.

Hippocampal and hypothalamic transcription of mRNA species for IL-1β, TNF-α and IL-6 was assessed using quantitative PCR after systemic challenge (i.p.) with saline, IL-1β (15 µg/kg) or TNF-α (50 µg/kg) at 1, 2, 4, 8 and 24 hours post-injection. All data groups were compared by two-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between each IL-1β group and the equivalent saline groups and comparing the cytokines directly. # denotes IL-1β/TNF-α treatment is significantly different to equivalent saline treatment at the time point indicated; * indicates significant difference between IL-1β and TNF-α. ^#/*p<0.05 # #/**p<0.01, ^{# # #/***}p<0.001. All data have been presented as mean±SEM, n=5 for all IL-1β and TNF-α treated groups except 2 h (n=4) and saline groups n≥3.

Figure 8. Impact of systemic IL-1β and TNF-α challenge on hippocampal cell activation and pathway markers at 1, 2, 4, 8 and 24 hours.

Hippocampal mRNA transcription of CCL2, CXCL1, PTX3 and uPAR was measured using quantitative PCR after systemic challenge (i.p.) with saline, IL-1β (15 µg/kg) or TNF-α at 1, 2, 4, 8 and 24 hours post-injection. All data groups were compared by two-way ANOVA, followed by Bonferroni post-hoc tests comparing differences between each IL-1β or TNF-α group and the equivalent saline groups and comparing the cytokines directly. # denotes treatment is significantly different to saline, while * denotes a difference between IL-1β and TNF-α. #/* p < 0.05, # #/** p < 0.01, # # #/*** p<0.001. All data have been presented as mean±SEM, n=5 for all IL-1β/TNF-α groups, except all 2 hour groups (n=4). All saline groups n≥4 except 4 and 24 hours n≥3.

Figure 9. Expression of pro-inflammatory mediators at the brain vasculature following peripheral immune challenge.

Representative micrographs of vascular NFκB p65, COX-2, CCL2 and CXCL1 2h after i.p. challenge with either saline, LPS (100µg/kg), IL-1β (15µg/kg) or TNF-α (50µg/kg). All pictures were taken from the hippocampus, except for COX-2, which were taken from the thalamus since neuropil labelling with COX-2 is also high in the hippocampus, thus reducing the contrast with activated endothelium. Scale bars are 20µm and 50µm as shown.

Figure 10. c-Fos expression in the central nucleus of the amygdala (CeA; A-D) and in the pial membrane (E–H) in brains of mice 2h after i.p. treatment with saline, LPS (100µg/kg), IL-1β (15µg/kg) or TNF-α (50µg/kg).

cFOS-positive neurons in the CeA are quantified and shown in the integrated table. ** denotes statistically significant difference from saline by Bonferroni post-hoc test after significant one way ANOVA.