Diesel exhaust particles disrupt blood-retina barrier integrity via TLR2 and TLR4 activation

Abstract

Diesel exhaust particles (DEPs), a major component of air pollution, are well-known to induce inflammation and vascular dysfunction. However, the molecular mechanisms linking DEP exposure to the disruption of the blood–retina barrier (BRB) remain poorly understood. Toll-like receptors (TLRs), particularly TLR2 and TLR4, play critical roles in inflammatory signaling and may contribute to DEP-induced retinal endothelial dysfunction. This study investigates the involvement of TLR2 and TLR4 in mediating DEP-induced disruption of the BRB and evaluates the protective effects of TLR inhibition using both in vitro and in vivo experiments. U937 human macrophages were exposed to DEPs of ultrafine size (＜0.2 μm), and the mRNA expression of TNF-α and IL-1β was quantified. Conditioned media from DEP-exposed U937 cultures were then used to treat human retinal endothelial cells (HRECs). DEP exposure significantly increased TNF-α and IL-1β mRNA expression in U937 macrophages. Conditioned media from DEP-exposed U937 macrophages reduced claudin-5 and ZO-1 expression in HRECs, resulting in increased BRB permeability. Inhibition of TLR2 and TLR4 using C29 and TAK242, respectively, significantly attenuated TNF-α and IL-1β mRNA expression in DEP-exposed U937 macrophages and preserved BRB integrity by maintaining claudin-5 and ZO-1 expression in HRECs. In the mouse model, DEP exposure caused a marked reduction in claudin-5 and ZO-1 levels in retinal vessels, whereas treatment with C29 and TAK242 mitigated the loss of these tight junction proteins. This study demonstrates that DEPs induce inflammation and BRB dysfunction through TLR2 and TLR4 activation, leading to increased vascular permeability and potential retinal damage. Furthermore, TLR2 and TLR4 inhibition may be a promising therapeutic strategy to protect retinal health from air pollution–induced damage. [BMB Reports 2025; 58(7): 300-306].

Fig. 1

TNF-α and IL-1β mRNA expression in U937 human macrophages at 6, 24, and 48 h after DEP exposure followed by washing. (A) Schematic representation of the experimental procedure. (B, C) TNF-α and IL-1β mRNA expression. TNF-α (B) and IL-1β (C) mRNA levels were significantly elevated at each time point following DEP exposure compared with those in the control. *P < 0.05 vs. control.

Fig. 2

Claudin-5 and ZO-1 expression and permeability in human retinal endothelial cells (HRECs) treated with conditioned media from U937 macrophages exposed to DEP. (A) Schematic representation of the experimental procedure. (B) Immunocytochemical staining. Claudin-5 and ZO-1 expression in HRECs was markedly reduced after treatment with conditioned media from DEP-exposed U937 macrophages. (C, D) Western blot analysis. Claudin-5 (C) and ZO-1 (D) protein levels in HRECs were significantly decreased following treatment with DEP-exposed U937-conditioned media compared with the control. (E) TEER permeability assay. Permeability in HRECs was significantly increased after treatment with DEP-exposed U937-conditioned media compared with the control. Scale bar = 30 μm, *P < 0.05 vs. control.

Fig. 3

Expression levels of TLR2/TLR4 and TNF-α/IL-1β mRNA in U937 macrophages exposed to DEPs with TLR2 (C29) or TLR4 (TAK242) inhibitors. (A) Schematic representation of the experimental procedure. (B, C) TLR2 and TLR4 expression levels. Compared with those in the control, TLR2 (B) and TLR4 (C) levels in U937 macrophages were significantly elevated following DEP exposure. TLR2 and TLR4 expression levels were significantly restored following treatment with C29 and TAK242 before DEP exposure, respectively. (D, E) TNF-α and IL-1β mRNA expression. TNF-α (D) and IL-1β (E) mRNA levels were significantly lower in U937 macrophages treated with C29 or TAK242 before DEP exposure than in cells that received DEP exposure alone without TLR inhibitors. *P < 0.05 vs. control, ^†P < 0.05 vs. DEP.

Fig. 4

Claudin-5 and ZO-1 expression and permeability in HRECs treated with conditioned media from U937 macrophages treated with C29 or TAK242 before exposure to DEPs. (A) Schematic representation of the experimental procedure. (B) Immunocytochemical staining. Claudin-5 and ZO-1 expression was markedly preserved in HRECs cultured in conditioned media from U937 macrophages treated with C29 or TAK242 before DEP exposure, than in HRECs that received DEP exposure alone without pretreatment with TLR inhibitors. (C, D) Western blot analysis. Claudin-5 (C) and ZO-1 (D) protein levels in HRECs were significantly restored following treatment with conditioned media from U937 macrophages treated with C29 or TAK242 before DEP exposure. (E) TEER permeability assay. Permeability was significantly lower in HRECs treated with conditioned media from U937 macrophages treated with C29 or TAK242 before DEP exposure, than in HRECs that received DEP exposure alone without pretreatment with TLR inhibitors. Scale bar = 30 μm, *P < 0.05 vs. control, ^†P < 0.05 vs. DEP.

Fig. 5

Claudin-5 and ZO-1 expression in retinal vessels of mice exposed to DEP and treated with C29 or TAK242. (A) Schematic representation of the experimental procedure. (B) Immunohistochemical staining. Claudin-5 and ZO-1 expression was markedly preserved in CD31⁺ retinal vessels of mice exposed to DEP with C29 or TAK242 than in those of mice exposed to DEP alone without TLR inhibitors. (C, D) Western blot analysis. Claudin-5 (B) and ZO-1 (C) protein levels in retinal lysates were significantly higher in mice that received DEP exposure with C29 or TAK242 than in those that received DEP exposure alone without TLR inhibitors. Scale bar = 50 μm, *P < 0.05 vs. control, ^†P < 0.05 vs. DEP (n = 3 per group).