NF-κB/NLRP3 Translational Inhibition by Nanoligomer Therapy Mitigates Ethanol and Advanced Age-Related Neuroinflammation

Abstract

Binge alcohol use is increasing among aged adults (>65 years). Alcohol-related toxicity in aged adults is associated with neurodegeneration, yet the molecular underpinnings of age-related sensitivity to alcohol are not well described. Studies utilizing rodent models of neurodegenerative disease reveal heightened activation of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Nod like receptor 3 (NLRP3) mediate microglia activation and associated neuronal injury. Our group, and others, have implicated hippocampal-resident microglia as key producers of inflammatory mediators, yet the link between inflammation and neurodegeneration has not been established in models of binge ethanol exposure and advanced age. Here, we report binge ethanol increased the proportion of NLRP3⁺ microglia in the hippocampus of aged (18-20 months) female C57BL/6N mice compared to young (3-4 months). In primary microglia, ethanol-induced expression of reactivity markers and NLRP3 inflammasome activation were more pronounced in microglia from aged mice compared to young. Making use of an NLRP3-specific inhibitor (OLT1177) and a novel brain-penetrant Nanoligomer that inhibits NF-κB and NLRP3 translation (SB_NI_112), we find ethanol-induced microglial reactivity can be attenuated by OLT1177 and SB_NI_112 in microglia from aged mice. In a model of intermittent binge ethanol exposure, SB_NI_112 prevented ethanol-mediated microglia reactivity, IL-1β production, and tau hyperphosphorylation in the hippocampus of aged mice. These data suggest early indicators of neurodegeneration occurring with advanced age and binge ethanol exposure are NF-κB- and NLRP3-dependent. Further investigation is warranted to explore the use of targeted immunosuppression via Nanoligomers to attenuate neuroinflammation after alcohol consumption in the aged.

Keywords: NLRP3 inflammasome; advanced aging; alcohol; microglia; neurodegeneration.

Figure 1.. Hippocampal Microglia from the Brains of Aged Mice Upregulate NLRP3 in Response to Binge Ethanol Compared to Young.

(A) Young (3–4 months old) and aged (18–20 months old) female C57BL/6N mice were exposed to intragastric gavages of ethanol (3 g/kg) or vehicle every other day for 10 total exposures. Multispectral imaging was performed on left hemispheres of young and aged ethanol and vehicle treated mice. Each brain hemisphere was imaged using a whole slice scanning system and representative images at 10x and 40x magnification are shown. (B) The proportion of microglia (Iba-1⁺ cells) that were NLRP3⁺ positive in CA3 of the hippocampus was quantified using QuPath software. n = 8–10 per group, means and SEM are reported. Values were significantly different from each other determined by 2-way ANOVA with Sidak’s post hoc test, * p < 0.05, **p < 0.01, *** p<0.001.

Figure 2.. Ex vivo Ethanol Exposure Potentiates Expression of NLRP3 and Microglia Activation Markers in Microglia from the Aged Mice Compared to Young.

Primary microglia were isolated from young and aged mice via density gradient. Cells were acclimated overnight with serum free media and challenged with or without 50 mM of ethanol for 24 hours. Cells were then fixed and processed for immunocytochemistry using antibodies against (A) CD68 and Iba1, (B) NLRP3 and TLR4, or (C) mature IL-1b. Representative images are shown at 20x objective. (D) Quantification of total staining intensity for each marker was performed using ImageJ and represented as staining intensity normalized to the number of DAPI⁺ positive cells. Individual data points are represented, n = 3–4 biological replicates per group. Means and SEM are reported, values were significantly different from each other as determined by 2-way ANOVA with Sidak’s post-hoc testing, * p < 0.05, ** p <0.01, *** p <0.001.

Figure 3.. NLRP3 Inhibition Reduces IL-1β Production and Indicators of Microglia Reactivity Due to Age and Ethanol Exposure Ex vivo.

Primary microglia from young and aged mice were isolated via density gradient. Cells were cultured overnight with serum free media and then treated with 10μM of OLT1177 or a vehicle. One hour later, cells were challenged with 50mM ethanol. Twenty-four hours following ethanol challenge, cells were fixed and assessed for microglia reactivity markers indicators of NLRP3 inflammasome activation via immunocytochemistry. (A) Iba-1 and mature IL-1β and (B) CD68 and cleaved caspase-1. (C) Quantification of staining intensity was performed using ImageJ and represented as staining intensity normalized to the number of DAPI⁺ positive cells. Individual data points are represented, n = 4–5 biological replicates per group. Values were significantly different from each other as determined by 2-way ANOVA with Sidak’s post-hoc testing, * p < 0.05.

Figure 4.. Suppression of NF-κB and NLRP3 mRNA Translation Normalizes IL-1β Production and Indicators of Microglia Reactivity Due to Age and Ethanol Exposure Ex vivo.

Primary microglia from young and aged mice were isolated via density gradient. Cells were cultured overnight with serum free media and then treated with 10μM of SB_NI_112 or a vehicle. One hour later, cells were challenged with 50mM ethanol. Twenty-four hours following ethanol challenge, cells were fixed and assessed for microglia reactivity markers indicators of NLRP3 inflammasome activation via immunocytochemistry. (A) Iba-1 and mature IL-1β and (B) CD68 and cleaved caspase-1. (C) Quantification of staining intensity was performed using ImageJ and represented as total staining intensity normalized to the number of DAPI⁺ positive cells. Individual data points are represented, n = 4–5 biological replicates per group. Values were significantly different from each other as determined by 2-way ANOVA with Sidak’s post-hoc testing, * p < 0.05, ** p <0.01.

Figure 5.. SB_NI_112 Mitigates Advanced Age and Ethanol-Related IL-1β Production in the Hippocampus.

(A) Aged (18–20 month-old) female mice were subjected to 10 intragastric gavages of ethanol (3g/kg) or vehicle every other day totaling 10 exposures. At the time of each gavage, animals were treated with SB_NI_112 (150mg/kg) or sterile 1x PBS via i.p. injection. Hippocampal tissue was isolated 18 hours after final exposure. (B) Paraffin-embedded mouse brain tissue was deparrafinized followed by immunohistochemistry with an antibody against mature IL-1β . (C) Expression of IL-1β staining in CA3 of the hippocampus from all treatment groups shown at 10x magnification. Insets show neuronal ribbon at 20x magnification, white arrow indicates IL-1β staining. Individual staining intensity values are represented, n = 4–9 per group. Values were significantly different from each other determined by 2-way ANOVA with Sidak’s post hoc test, * p < 0.05, ** p <0.01.

Figure 6.. Ethanol Related Increases in Microglia Reactivity Markers are Reduced by NF-κB and NLRP3 mRNA Suppression in the Hippocampus of Aged Mice.

Aged female mice were subjected to our intermittent binge ethanol exposure paradigm, consisting of 3g/kg ethanol or vehicle gavages every-other day, totaling 10 exposures. Animals were treated with i.p. injections of SB_NI_112 (150mg/kg) or sterile 1x PBS at the time of gavage. (A) Paraffin-embedded mouse brain tissue was deparrafinized followed by immunohistochemistry with an antibody against microglia reactivity marker Iba-1. Expression of Iba-1 staining in CA3 of the hippocampus from all treatment groups with representative images of microglia morphology shown to the right of each image at 20x magnification. (B) Quantification of Iba-1 staining intensity and average microglia soma area. Individual values are represented, n = 4–9 per group. Values were significantly different from each other determined by 2-way ANOVA with Sidak’s post hoc test, * p < 0.05 ** p < 0.01.

Figure 7.. Hippocampal Tau Hyperphosphorylation Due to Advanced Age and Ethanol Exposure are Alleviated by SB_NI_112.

Aged female mice were exposed to 10 gavages of 3g/kg ethanol or vehicle every-other day, with or without SB_NI_112 treatment. Paraffin-embedded mouse brain tissue was deparrafinized followed by immunodetection (A) tau phosphorylation site T231. Counterstaining was performed to visualize nuclei with hematoxylin. Individual values are represented, n = 4–9 per group. Values were significantly different from each other as determined by 2-way ANOVA with Sidak’s post-hoc testing, ** p <0.01.