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. 2023 Jun 6:4:100107.
doi: 10.1016/j.crtox.2023.100107. eCollection 2023.

Evaluating the effect of acute diesel exhaust particle exposure on P-glycoprotein efflux transporter in the blood-brain barrier co-cultured with microglia

Grace V Aquino  1 , Amjad Dabi  2 , Gabriel J Odom  3 , Ramon Lavado  1 , Kaitlin Nunn  1 , Kathryn Thomas  1 , Bennett Schackmuth  1 , Nazeel Shariff  1 , Manogna Jarajapu  1 , Morgan Pluto  1 , Sara R Miller  1 , Leah Eller  1 , Justin Pressley  1 , Rishi R Patel  1 , Jeffrey Black  1 , Erica D Bruce  1
Affiliations

Evaluating the effect of acute diesel exhaust particle exposure on P-glycoprotein efflux transporter in the blood-brain barrier co-cultured with microglia

Grace V Aquino et al. Curr Res Toxicol. .

Abstract

A growing public health concern, chronic Diesel Exhaust Particle (DEP) exposure is a heavy risk factor for the development of neurodegenerative diseases like Alzheimer's (AD). Considered the brain's first line of defense, the Blood-Brain Barrier (BBB) and perivascular microglia work in tandem to protect the brain from circulating neurotoxic molecules like DEP. Importantly, there is a strong association between AD and BBB dysfunction, particularly in the Aβ transporter and multidrug resistant pump, P-glycoprotein (P-gp). However, the response of this efflux transporter is not well understood in the context of environmental exposures, such as to DEP. Moreover, microglia are seldom included in in vitro BBB models, despite their significance in neurovascular health and disease. Therefore, the goal of this study was to evaluate the effect of acute (24 hr.) DEP exposure (2000 μg/ml) on P-gp expression and function, paracellular permeability, and inflammation profiles of the human in vitro BBB model (hCMEC/D3) with and without microglia (hMC3). Our results suggested that DEP exposure can decrease both the expression and function of P-gp in the BBB, and corroborated that DEP exposure impairs BBB integrity (i.e. increased permeability), a response that was significantly worsened by the influence of microglia in co-culture. Interestingly, DEP exposure seemed to produce atypical inflammation profiles and an unexpected general downregulation in inflammatory markers in both the monoculture and co-culture, which differentially expressed IL-1β and GM-CSF. Interestingly, the microglia in co-culture did not appear to influence the response of the BBB, save in the permeability assay, where it worsened the BBB's response. Overall, our study is important because it is the first (to our knowledge) to investigate the effect of acute DEP exposure on P-gp in the in vitro human BBB, while also investigating the influence of microglia on the BBB's responses to this environmental chemical.

Keywords: Amyloid-beta; Blood–brain barrier; Diesel exhaust particles; Inflammation; Microglia; P-glycoprotein.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

None
Graphical abstract
Fig. 1
Fig. 1
The Role of P-glycoprotein and other Key Aβ Transporters in the Soluble Aβ Clearance Pathways of the BBB. Soluble Aβ in the brain parenchyma can undergo two basic fates: (1) aggregation into insoluble, neurotoxic Aβ aggregates (plaques) in the interneuron space, and (2) extrusion from the CNS across the BBB or via cerebrospinal fluid (CSF). Soluble Aβ can be eliminated from the brain via two main pathways: (1) enzymatic degradation and (2) receptor mediated clearance. In the first case, soluble Aβ is phagocytosed and then degraded by activated microglia (CNS macrophages) or peptidases (P) like Insulin-Degrading Enzyme (IDE), Neprilysin (NEP), Endothelin-Converting Enzyme (ECE), Angiotensin-Converting Enzyme (ACE), Matrix Metalloproteinases (MMPs), etc. In the second case, soluble Aβ is transported across the BBB and exported out of the CNS into systemic circulation through direct binding to P-gp and LRP-1 transporters, or through first binding Aβ chaperones (C) like apolipoprotein E (ApoE), apolipoprotein J (ApoJ), albumin, and α2-macrogobulin (α2M) that then deliver Aβ peptide to these efflux transporters. Importantly, P-gp is the most clinically-relevant multidrug resistant pump that besides extruding Aβ peptide, also extrudes xenobiotics and small molecule drugs (SMD), effectively limiting their accumulation in the CNS. Once in systemic circulation, Aβ can (a) re-enter the CNS via the RAGE receptor, (b) be sequestered by soluble LRP (sLRP), which acts as the main Aβ sink, and/or (c) be transported by chaperone molecules (ApoE and α2M) to the liver and kidneys for elimination from the body.
Fig. 2
Fig. 2
Relative Gene Expression of P-gp after 24 hr. DEP exposure in monoculture and co-culture. RT-qPCR was used to assess the relative gene expression of P-gp (MDR1) in the endothelial monoculture and the endothelial-microglial co-culture after 24 hr. exposure to 2 mg/ml DEP. Bars represent mean fold change from control 2–ΔΔCT normalized with SDHA and expressed as a percentage of the unexposed control ± SEM. Statistical significance was considered p ≤ 0.05 (N ≥ 3, Mann-Whitney U Test). No statistical significance was detected.
Fig. 3
Fig. 3
P-gp function after 24 hr DEP exposure in monoculture and co-culture. The Rhodamine-123 Accumulation Assay was used to assess the transport activity or function of P-gp in the endothelial monoculture and the endothelial-microglial co-culture after 24 hr exposure to 2 mg/ml DEP. Bars represent mean fold change relative to the control ± SEM. Statistical significance was considered p ≤ 0.05 (N ≥ 3, Mann-Whitney U Test). No statistical significance was detected.
Fig. 4
Fig. 4
A. TEER development in BBB models over time and after DEP exposure. Monoculture and co-culture BBB models were developed following a modified method from Förster et al, and their TEER were measured daily for 6 days. When the BBB models reached peek TEER on day 4, the models were exposed luminally to 2 mg/ml DEP or control media for 24 hr. Lines represent mean TEER ± SEM. B. Change in TEER after 24 hr. DEP exposure in monoculture and co-culture. Monoculture and co-culture BBB models were exposed to 2 mg/ml DEP for 24 hr. Bars represent mean fold change relative to the control ± SEM. Statistical significance was considered p ≤ 0.05 (N ≥ 3, Mann-Whitney U Test). Asterisk denotes significance from unexposed control. Black dot denotes significance from DEP-exposed monoculture.
Fig. 5
Fig. 5
Inflammatory marker profiles of monoculture and co-culture BBB models after 24 hr DEP exposure. Luminex bead immunoassays were used to measure 10 cytokines/chemokines in the endothelial monoculture and the endothelial-microglial co-culture after 24 hr exposure to 2 mg/ml DEP. Bars represent mean fold change from unexposed controls ± SEM. Statistical significance was considered p ≤ 0.05 (N = 4, Mann-Whitney U Test). No statistical significance was detected.
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