Microglial TLR4 mediates white matter injury in a combined model of diesel exhaust exposure and cerebral hypoperfusion

Abstract

Background:

Air pollution particulate matter (PM) exposure and chronic cerebral hypoperfusion (CCH) contribute to white matter toxicity through shared mechanisms of neuroinflammation, oxidative stress, and myelin breakdown. Prior studies showed that exposure of mice to joint PM and CCH caused supra-additive injury to corpus callosum white matter. This study examines the role of toll-like receptor 4 (TLR4) signaling in mediating neurotoxicity and myelin damage observed in joint PM and CCH exposures.

Methods:

Experiments utilized a novel murine model of inducible monocyte/microglia-specific TLR4 knockout (i-mTLR4-ko). Bilateral carotid artery stenosis (BCAS) was induced surgically to model CCH. TLR4-intact (control) and i-mTLR4-ko mice were exposed to 8 weeks of either aerosolized diesel exhaust particulate (DEP) or filtered air (FA) in eight experimental groups: 1) control/FA (n=10), 2) control/DEP (n=10), 3) control/FA+BCAS (n=9), 4) control/DEP+BCAS (n=10), 5) i-mTLR4-ko/FA (n=9), 6) i-mTLR4-ko/DEP (n=8), 7) i-mTLR4-ko/FA+BCAS (n=8), and 8) i-mTLR4-ko/DEP+BCAS (n=10). Corpus callosum levels of 4-hydroxynonenal (4-HNE), 8-Oxo-2’-deoxyguanosine (8-OHdG), ionized calcium-binding adapter molecule 1 (Iba-1), and degraded myelin basic protein (dMBP) were assayed via immunofluorescence to measure oxidative stress, neuroinflammation, and myelin breakdown, respectively.

Results:

Compared with control/FA mice, control/DEP+BCAS mice exhibited increased dMBP (41%; p<0.01), Iba-1 (51%; p<0.0001), 4-HNE (100%; p<0.0001), and 8-OhdG (65%; p<0.05). I-mTLR4 knockout attenuated responses to DEP/BCAS for all markers.

Conclusions:

i-mTLR4-ko markedly reduced neuroinflammation and oxidative stress and attenuated white matter degradation following DEP and CCH exposure. This suggests a potential role for targeting TLR4 signaling in individuals with vascular cognitive impairment, particularly those exposed to substantial ambient air pollution.

Graphical Abstract

Introduction:

Chronic cerebral hypoperfusion (CCH) secondary to carotid artery disease is a significant contributor to vascular dementia.¹ CCH increases blood-brain barrier permeability, rendering the brain susceptible to environmental neurotoxins including air pollution particulate matter (PM).² CCH and PM damage the brain via overlapping mechanisms including oxidative damage, neuroinflammation, and myelin degradation.^3,4 We previously demonstrated that joint nanoscale PM exposure and CCH in mice resulted in synergistic myelin toxicity.⁵

Toll-like receptor 4 (TLR4) signaling is implicated in both cerebral ischemia and PM-induced neurotoxicity.^6,7 TLR4 signaling increases following nanoscale PM exposure in vitro and in vivo, and TLR4 mediates neuroinflammation, oxidative damage and myelin breakdown in cerebral ischemia.^6,7,8

The present study utilizes a novel murine model with inducible macrophage/microglia-specific TLR4 knockout (i-mTLR4-ko) to examine the role of TLR4 signaling in white matter injury secondary to diesel exhaust particulate (DEP) and CCH exposures.

Methods

The principal investigator had access to all data and assumes responsibility for data integrity. Data are available from the corresponding author upon request.

Animals:

Animal handling, husbandry and euthanasia (Supplemental Methods) were performed according to University of Southern California Institutional Animal Care and Use Committee regulations and adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. ARRIVE guidelines were met (Supplemental Materials).

i-mTLR4-ko mice were generated using a Cre recombinase- loxP system.⁹ A Cx3cr1^CreER (JAX020940) mouse was crossed with a Tlr4^fl (JAX024872) mouse. To induce TLR4 deficiency, tamoxifen (20mg/ml) mixed in corn oil (150mg/kg) or corn oil alone (control) was administered intraperitonially 12 and 10 days before DEP exposure initiation. This timing allows peripheral macrophage turnover prior to experiment initiation while ensuring that microglia exhibit TLR4 knockout throughout the exposure duration.¹⁰

Diesel Exhaust Exposure:

Male mice were exposed to 200 hours (5 hours/day, 5 days/week for 8 weeks) of aerosolized DEP (NIST SRM 2975) or filtered air (FA). Details are described in Supplemental Methods.

Bilateral Carotid Artery Stenosis (BCAS) Surgery:

Either BCAS, achieved via application of a microcoil around each common carotid artery, or no surgery was performed 30 days prior to the end of DEP exposure. Details are described in Supplemental Methods.

Immunofluorescence Staining:

Formalin-fixed, paraffin-embedded brains were sectioned coronally. Anterior corpus callosum 5μm sections were stained for 4-hydroxynonenal (4-HNE); 8-Oxo-2’-deoxyguanosine (8-OHdG); ionized calcium binding adaptor molecule 1 (Iba-1); and degraded myelin basic protein (dMBP). Representative images are displayed in Supplemental Figure 1. See Supplemental Methods and Supplemental Table 1 for more information.

Statistical Analyses:

Eight experimental groups were compared: 1) control/FA (n=10), 2) control/DEP (n=10), 3) control/FA+BCAS (n=9), 4) control/DEP+BCAS (n=10), 5) i-mTLR4-ko/FA (n=9), 6) i-mTLR4-ko/DEP (n=8), 7) i-mTLR4-ko/FA+BCAS (n=8), and 8) i-mTLR4-ko/DEP+BCAS (n=10). Integrated fluorescent density measurements were compared across groups utilizing one-way ANOVA with Tukey’s test for post-hoc analysis. Statistical significance was defined at two-sided alpha of 0.05. See Supplemental Methods for more information. Significant comparisons relevant to the study hypothesis/outcomes are presented in Figure 1; complete ANOVA results are displayed in Supplemental Tables 2–5.

Figure 1: Corpus Callosum Immunofluorescence.

Graph depicts immunofluorescence integrated density levels (A. 4-HNE; B. 8-OHdG; C. Iba-1; D. dMBP). Data were analyzed using one-way ANOVA with Tukey’s test, with significance defined at two-sided alpha of 0.05. Significant comparisons relevant to study hypothesis/outcomes are displayed; Supplemental Tables 2–5 present complete ANOVA results. * p<0.05, ** p<0.01, **** p<0.0001. FA, filtered air; i-mTLR4-ko, inducible macrophage/microglial TLR4 knockout; DEP, diesel exhaust particulate; BCAS, bilateral carotid artery stenosis.

Results

Corpus Callosum Immunofluorescence

TLR4 Intact Controls:

DEP treatment alone significantly increased 4-HNE (P<0.01; Figure 1A). There were nonsignificant increases in 8-OHdG, Iba-1, and dMBP. Control/ FA+BCAS mice exhibited significant elevations in Iba-1 (P<0.05; Figure 1C) and nonsignificant elevations in 4-HNE, 8-OHdG, or dMBP compared with control/FA mice. Control/DEP+BCAS mice demonstrated elevated 4-HNE (100%; P<0.0001; Figure 1A), 8-OHdG (65%; P<0.05; Figure 1B), Iba-1 (51%; P<0.0001; Figure 1C), and dMBP (41%; P<0.01; Figure 1D) above control/FA mice.

TLR4 deletion:

i-mTLR4-ko/FA mice did not exhibit differences in 4-HNE, 8-OHdG, Iba-1, and dMBP compared to TLR4-intact controls (Fig.1, A–D). i-mTLR4-ko blocked elevations in 4-HNE in response to DEP exposure. i-mTLR4-ko prevented significant elevations in Iba-1 after BCAS exposure. i-mTLR4-ko prevented significant rises in 4-HNE, 8-OHdG, Iba-1 and dMBP following DEP+BCAS exposure. Finally, i-mTLR4-ko/DEP+BCAS mice demonstrated 90% lower 4-HNE (p<0.0001), 23% lower Iba-1 (p<0.05), and 38% lower dMBP (p<0.01) than control/DEP+BCAS mice.

Discussion:

This study utilized a novel transgenic TLR4 knockout mouse to demonstrate the importance of microglial TLR4 signaling in mediating white matter toxicity secondary to air pollution exposure and cerebral ischemia. Importantly, TLR4 deficiency decreased myelin degradation following DEP+BCAS exposure such that corpus callosum dMBP levels in i-mTLR4-ko/DEP+BCAS mice were comparable to levels in control/FA mice. TLR4 deficiency also decreased neuroinflammation and oxidative stress, corroborating previous studies investigating TLR4 signaling in cerebrovascular disease and air pollution exposure.^7,11,12

A potential confounding factor in this study is that CX3CR1 is expressed in peripheral macrophages as well as microglia, which could result in broader TLR4 deletion and systemically reduced inflammation in tamoxifen-administered mice. However, frequent turnover of peripheral macrophages renders this issue less likely in an eight-week exposure. The use of only young, male mice is an additional study limitation; future investigations of TLR4 knockout in females and older mice will be useful to evaluate age and sex differences in the TLR4-mediated response to DEP and CCH exposures.

Chronic exposure to ambient particulate matter is associated with cognitive decline in humans.^13,14 We previously demonstrated that, while nanoscale PM exposure alone did not disrupt working memory, combined nanoscale PM inhalation and BCAS in a murine model led to more substantial memory impairment than BCAS alone.⁵ The present study lacks behavioral data to determine the effects of TLR4 knockout on cognitive function; this avenue would benefit from further investigation.

Conclusions:

We present evidence that microglial TLR4 knockout prevents joint DEP- and CCH-mediated white matter toxicity in a murine model. Targeting TLR4 signaling may potentially be a viable therapeutic strategy to mitigate myelin degradation and cognitive decline in individuals with significant ambient air pollution exposure and coexisting vascular disease.

Non-standard Abbreviations and Acronyms:

CCH

chronic cerebral hypoperfusion

PM

particulate matter

BCAS

bilateral carotid artery stenosis

i-mTLR4-ko

inducible macrophage/microglia-specific toll-like receptor 4 knockout

DEP

diesel exhaust particulate

FA

filtered air