Since glomerular endothelial cells (GECs), the glomerular basement membrane, and podocytes are believed to contribute to the glomerular filtration barrier, damages to any of these intrinsic cell types reportedly lead to the development of glomerulonephritis (GN) [1]. In clinical practice, it is well known that viral infections can trigger the development of inflammatory GN or the worsening of pre-existing GN [2]. Recognition of the molecular pattern of viral pathogens by Toll-like receptor 3 (TLR3) found in intracellular endosomes and subsequent immunoreactions is important in host antiviral defenses [3,4]. TLR3 signaling cascades activate the TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent pathways via transcriptional factors, i.e., nuclear factor κB (NF-κB) and interferon regulatory factor 3 (IRF3), and the subsequent release of inflammatory chemokines, cytokines, adhesion molecules, and finally type I interferons (IFN) [3]. Notably, the activation of TLR3 and their downstream immune responses can be induced not only by infectious pathogens but also non-infectious stimulation such as endogenous ligands [3]. It has also been reported that the expression of TLR3 in residual glomerular cells is confirmed in biopsy specimens from patients with GN [2,5]. Thus, regional viral and “pseudo” viral immunoreactions via the activation of TLR3 signaling in the intrinsic glomerular cells have been postulated to be involved in the pathogenesis of some GN [2,6]. Interestingly, sustained activation of type I IFN is thought to be involved in the pathogenesis of lupus nephritis (LN) [6,7]. Therefore, the involvement of the TLR3 signaling system in the intrinsic glomerular cells is postulated at least in part in the pathogenesis of LN [6,8-10].

It has been reported that GECs express various adhesion molecules in response to proinflammatory cytokines and play a role in the pathogenesis of hemodynamic disturbances [11]. Furthermore, GECs are directly exposed to circulating viral particles in the glomerulus; therefore, endothelial antiviral and “pseudo” viral immunoreactions through TLR3 signaling are thought to play a role in the initiation of glomerular inflammation [12,13]. Thus far, considering the implications of TLR3 signaling in the pathogenesis of LN, we treated human GECs with polyinosinic-polycytidylic acid (poly IC), an authentic viral double-stranded RNA, and then examined the expressions of representative neutrophil chemoattractant C-X-C motif chemokine 1 (CXCL1)/GROα, the adhesion molecule E-selectin, and macrophage chemoattractant CX3CL1/fractalkine [13,14]. We found that the activation of TLR3 induced the endothelial expression of CXCL1, E-selectin, and CX3CL1, and that these involved TLR3 signaling cascades of NF-κB, IRF3, and IFN-β (Figure 1). Intense endothelial CXCL1 expression was observed in biopsy specimens from patients with crescentic GN [13]. Given that E-selectin, CXCL1 and CX3CL1 in GECs contribute to regional neutrophil and macrophage recruitment, which leads to the development of GN, TLR3 signaling in GECs may control initial inflammation in the glomerulus [13,14]. Further, we found that poly IC increased plasminogen activator inhibitor-1 (PAI-1) and interleukin-6 (IL-6) expressions in GECs [15,16]. Since PAI-1 inhibits fibrinolysis, an imbalance of activity between PAI-1 and tissue plasminogen activator contributes to glomerulosclerosis [17], endothelial TLR3 signaling is also involved in the chronic lesion of GN. Interestingly, cytosolic sensors of viral RNA, retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated gene 5 were found to be involved in IL-6 production via TLR3 signaling in GECs [16]. However, endothelial PAI-I expression did not depend on these cytosolic recognition sensors [15]. Thus, the expression of inflammatory molecules through TLR3 signaling in human GECs may be tightly regulated, although this theory is yet to be elucidated.

Despite some limitations, possible involvement of endothelial TLR3 signaling, which is associated with the continuous activation of type I IFN as well as a regional expression of various inflammatory molecules, is thought to be involved in the pathogenesis of GN, particularly in LN [6,7].

Although the European League against Rheumatism and the European Renal Association-European Dialysis and Transplant Association (EULAR/ERA-EDTA) recommended the use of an antimalarial agent, chloroquine for patients with LN [18], the detailed beneficial effects of chloroquine against glomerular inflammation is yet to be elucidated. Previously, we examined the effects of chloroquine on the representative TLR3/IFN-β-signaling axis, TLR3/IFN-β/RIG-I/chemokine (C-C motif) ligand 5 (CCL5) in human glomerular mesangial cells treated with poly IC and found that 10 μg/mL chloroquine attenuates mesangial TLR3 signaling in the early phase of NF-κB activation [19]. Subsequently, we examined the effect of chloroquine on the endothelial TLR3 signaling and found that pretreating cells with chloroquine but not dexasamethasone attenuated poly IC-induced CX3CL1 expression in GECs [14]. Further, we found that pretreating cells with chloroquine, and also hydroxychloroquine, but not dexamethasone, attenuated poly IC-induced PAI-1 expression in GECs [15]. Our results may further support regional renoprotective effects of the antimalarial agents, chloroquine in treating LN [14,15]. More detailed studies are required to confirm our preliminary findings.

In daily clinical practice, it has been reported that the minimum hydroxychloroquine target level to prevent flares in 171 lupus patients was, at least, more than 600 ng/mL [20]. Considering the blood concentration level, 10 μg/mL of chloroquine used in our experiments may be rather higher than postulated in this paper. However, the adequate blood level of the drug in patients with LN is yet to be determined. In addition, it is unclear whether adequate blood levels of the drug prevent activated TLR3 signaling in GECs. This issue should be clarified in future studies.

The activation of the TLR3 signaling cascade in residual renal cells and cross- talk of infiltrating monocytes, neutrophils, and lyomphocytes results in inducing type I IFNs release, which may be involved in the pathogenesis of LN [6,8,9]. Studies of drugs that have been useful in controlling glomerular inflammatory pathways mediated by the innate immunity are now underway.

There are no conflicts of interest to declare.

This study was supported by Grants-in-Aid of the Japan Society for Promotion of Science (JSPS KAKENHI Grant number: 16K10055 to H. T.).