In this study we brought evidence that HCE and HCF cells constitutively express mRNA specific for TLR2-4, TLR7 and TLR9 as well as TLR3- and TLR4- proteins on the cell surface. Our results suggest that these cells are capable of responding to TLR stimulation, culminating in the secretion of several pro-inflammatory mediators that are known to be down-stream effectors of the TLR pathway, along with up-regulation of their respective genes.
Our results show that while stimulation of HCE cells with LPS culminated in increased mRNA contents of both TLR3 and TLR4, HCE cells’ activation with poly I:C elicited increase mRNA levels of TLR3 only. This apparent inconsistency might be explained by a possible difference in the kinetics and half-lives of the different RNAs. A real-time PCR analysis conducted over a longer time-frame might counteract these differences.
Interestingly, activation of HCE TLR3 and TLR4 by poly I:C and LPS, while stimulating secretion of pro-inflammatory cytokines and the up-regulation of their respective genes, was found to decrease TLRs surface expression, as measured using flow cytometry. LPS-induced TLR4 suppression is an established concept. It has been described since the 1940s, that low doses of LPS can suppress the immune response to endotoxin challenge, a phenomenon termed LPS hyporesponsiveness or LPS tolerance [17–19]. The discovery of the Toll-like receptor family set a new interest in this phenomenon, and several mechanisms have been proposed to explain it, among them a decrease in surface expression of TLR4. The LPS tolerance might lead to better outcomes in patients with septic shock, perhaps by inhibiting the detrimental effects in an unchecked inflammatory process [20–22] Tolerance induction following TLR activation was previously observed in the human cornea. Kumar et al. observed that pre-exposure of HCE cells to low-dose flagellin, the ligand of TLR5, induced a state of tolerance, characterized by impaired functionality of the NF-κB, p38 and JNK pathways .
We hypothesize that the decrease in TLR3 and TLR4 surface expression in HCE cells following ligand activation is a generalization of the LPS tolerance described in the previous paragraph, and constitutes a negative-feedback loop that is important in containing the immune response, and might play a role in forming an immune-silent environment in the human cornea.
We observed that stimulation of TLR3 and TLR4 was followed by an increase in the content of TLR-specific mRNA. Interestingly, in apparent dissociation, the receptors’ protein content was noted to decrease. Our present findings are in accordance with a previous work on TLR4 expression in patients with sepsis, that revealed early TLR4-protein down-regulation, using flow cytometry. Skinner et al. postulated that these patients may at first respond with TLR4 activation and up-regulation, as shown by cytokine secretion profile, thereby over-expressing the pro-inflammatory cytokines that are characteristic of sepsis . A subsequent TLR4 down-regulation could reflect a possible negative feedback mechanism, important for mitigating the inflammatory process. We postulate that a similar mechanism is found in HCE cells of the human eye. It is reasonable to assume, that while inappropriate secretion of pro-inflammatory cytokines is at best to be avoided and thus silenced, upon stimulation of TLR3 and TLR4, a preparatory phase, involving enhanced transcription of TLR3 and TLR4-specific genes, is beneficent. We must stress, nonetheless, that this last proposition is a mere hypothesis that has to be investigated in a further work.
Stimulation of TLR3 and TLR4 in HCF did not elicit a statistically significant decrease in surface expression, as was noted in HCE cells. It is interesting to note, that unlike HCE cells that responded with profound secretion of pro-inflammatory cytokines following TLR activation, HCF secreted only IL-6 and IL-8. These findings might reflect a distinct behavior of HCF, which may be more resistant to TLR stimulation, and less prone to initiate a profound inflammatory response.