In this study, we found that the autophagy process was activated in DCs from active BD patients. Autophagic degradation was however decreased, which, in turn, results in proinflammatory cytokine release by DCs. Moreover, we found that miR-155 and its target protein TAB2 were involved in the proinflammatory cytokine production of DCs by regulating autophagy through the Akt/mTOR signaling pathway. These results indicate that miR-155 might take part in the development of BD by controlling autophagy. In other words our findings indicate that certain triggers in a genetically predisposed individual stimulates autophagosome formation, whereby a defective control of the subsequent degradation of autophagic products leads to a local stimulation of proinflammatory cytokine release.
It has been reported that autophagy was associated with the development of animal uveitis models.[30, 31] However, the role of autophagy in the pathogenesis of human uveitis is still unknown. In this study, we first detected the activity level of autophagy in DCs from active and inactive BD patients. The results showed that the autophagosomes were found in DCs from active BD patients. Furthermore, we found that as compared with inactive BD patients and healthy controls, the protein levels of two important markers of autophagy activity, Beclin-1 and LC3-Ⅱ, were significantly increased in DCs derived from active BD patients. Additionally, the protein expression level of P62 was significantly increased in DCs of active BD patients as compared with inactive BD patients and healthy controls, which indicated that there is an increased number of autophagosomes but a concurrent buildup of autophagic degradation products.
In view of the impaired autophagic flux in DCs of active BD patients, we further examined if the activity of autophagy is associated with the cytokine production by DCs. Our results showed that compared with healthy controls, the production of TNF-α, IL-6 and IL-1β by DCs from active BD patients stimulated with LPS were significantly increased. We also showed that the autophagy inhibitor (3-MA) or promoter (rapamycin) could upregulate or downregulate the production of TNF-α, IL-6 and IL-1β. These results proved that the autophagy activity level correlated with the production of cytokines by DCs. These results are consistent with previous studies showing that IL-1β, IL-17 andIL-18 levels were downregulated in murine DCs after autophagy was inhibited. Others showed that the production of IL-6, IFN-β and TNF-α by DCs were decreased after Beclin-1 was knocked out. These results indicate that a defective control of autophagy may trigger cytokine production by DCs.
Earlier we reported that the expression of miR-155 was downregulated in DCs obtained from active BD patients and was involved in cytokine production by DCs. It has also been reported that the expression of miR-155 was increased or decreased in various diseases and was involved in the development of these diseases by regulating autophagy.[17, 19, 34, 35] In view of the aforementioned reports, we next investigated whether miR-155 exerted its effects on cytokine production by DCs via the process of autophagy. To answer this question, an experiment with miR-155 mimic transfection was performed. The results showed that an miR-155 mimic could promote the expression of LC3-Ⅱ and Beclin-1, which meant that autophagy activity was increased. More importantly, the protein level of P62 was downregulated in DCs after miR-155 mimic was transfected, showing that autophagic degradation was increased. Additionally, miR-155 mimic transfection reduced the production of TNF-α, IL-1β and IL-6. The autophagy inhibitor, 3-MA, was able to counteract the effects of miR-155 on cytokine production. All these results suggested that autophagy was involved in the effects of miR-155 on cytokine production by DCs.
TAB2 has been reported to be the target of miR-155 and its protein expression level was elevated in DCs derived from active BD patients. It also been reported that TAB2 could bind to Beclin1 or ATG13 to regulate autophagy.[36-38] A further experiment was performed to investigate whether miR-155 regulates the process of autophagy through TAB2. The results showed that downregulation of TAB2 could promote the autophagic flux. The expression of TNF-α, IL-6 and IL-1β were decreased after TAB2 was downregulated. Taken together, these data indicate that miR-155 regulates autophagy by controlling the expression of TAB2. Our results are in agreement with previous studies that showed that miR-155 promoted autophagy via a decrease in TAB2 expression thereby stimulating osteoclast formation.
The Akt/mTOR pathway is one of the most common signaling pathways involved in the control of autophagy.[39, 40] Rapamycin, as the inhibitor of mTOR, has been shown to alleviate inflammation of the retina. It has also been reported that the use of this mTOR inhibitor was safe and effective in treating non-infectious uveitis.[42-44] In this study, we found that miR-155 could downregulate the phosphorylation level of mTOR and Akt in DCs.
The limitation of our research is that we did not investigate the function of autophagy in other types of uveitis and the detailed mechanisms on how a decreased autophagic degradation results in increased cytokine production. Further experiments are needed to explore the effects of treatment on the function of autophagy in DCs and if the dysfunction of autophagy is a common phenomenon of uveitis.