The present study demonstrates the involvement of the complement system in stem cell-mediated neuroregeneration under inflammatory conditions. We explored the regenerative ability and the role of C5L2 on BDNF secretion in DPSCs. We evaluated the C5L2 siRNA-treated BDNF production in DPSCs with or without LPS and found that C5L2 silencing could enhance BDNF production via the p38α pathway suggesting that C5L2 negatively modulates BDNF production in DPSCs.
The complement system is one of the most critical components of innate immunity which can be activated during apoptosis, necrosis and bacterial or viral pathogenesis 23–25. Besides its role in immunity, it facilitates the process of regeneration e.g., it has been reported to be involved in the regeneration of bone, liver and cardiac tissues 11–13. Further, Bergmann et al, have reported the linkage between inflammation and tissue regeneration 26. The main effect of the complement system seems to be mediated by its active fragment C5a and its receptor C5aR. While another C5a-like receptor 2 (C5L2) received less attention and its biological functions remained inscrutable 18,27. Unlike most other G-protein coupled receptors (GPCRs), C5L2 is deficient in G-protein coupling, yet; C5L2 in an emerging functional receptor to be known for its roles in inflammation and regeneration 28. In our study, we used LPS-stimulated DPSCs and evaluated C5L2-mediated BDNF production which suggests a positive role in enhanced production of this indispensable neurotrophin.
Previously, we demonstrated the role of complement system in odontogenic differentiation and neural regeneration using pulp fibroblasts and suggested that complement receptors i.e., C5aR and C5L2 facilitate dentin repair through DMP-1 expression and nerve fibers growth via increased BDNF secretion, respectively19–21. Recently, we studied C5aR-mediated BDNF production in DPSCs compared to bone marrow-derived mesenchymal stem cells (BM-MSCs) and concluded that DPSCs are a better choice for BDNF secretions than other stem cells when it comes to stem cell therapy in regenerative medicine17. In consistent with our study, Pagella et al. demonstrated that DPSCs are superior at enhancing nerve outgrowth via increased BDNF production compared to BM-MSCs 29. Another study showed that dental pulp-derived cells can differentiate into Schwann-like cells and secrete neurotrophins such as BDNF and NGF30. Several studies have established the capability of DPSCs to differentiate into neuronal cells and express neuronal markers 2,31,32. It is believed that the neural crest origin of DPSCs makes them an ideal choice for stem cell therapy, especially for neural regeneration. Considering several advantages of choosing DPSCs over other stem cells, we procured DPSCs for our study to evaluate the role of C5L2 in BDNF production.
Tissue regeneration following trauma and injury occurs in an inflammatory context. In this regard, we evaluated the effect of inflammation using LPS, which is one of the most potent inflammation inducers. We have previously established that lipoteichoic acid (LTA) stimulated dental pulp fibroblasts secrete higher levels of BDNF and enhanced axonal growth via C5aR modulation while C5aR antagonist abolished this potentiating effect and decreased axonal growth and length 16. Consistent with this, LPS treatment enhanced BDNF production in C5L2 silenced DPSCs suggesting a positive role of inflammation in DPSC-mediated tissue regeneration. We showed earlier that C5L2 silencing enhanced nerve outgrowth in dental pulp fibroblasts by increased BDNF secretion 19,20 and C5aR mediated nerve growth 33 suggesting a positive role of C5aR. Here, we studied the role of C5L2 siRNA silencing in BDNF secretion and our results showed increased BDNF production in C5L2 silenced DPSCs, which indicates a negative role of C5L2. Taken together, our results including our previous studies19,20 suggest that C5aR activity can be enhanced by C5L2 inhibition. The alternative approach of targeting C5L2 could provide an innovative therapeutic strategy, i.e., the possibility to enhance the positive action of C5aR in stem cell engineering and tissue regeneration by blocking the “inactive” C5L2 receptor.
The p38MAPK molecule is highly expressed in the brain and is known to be involved in several biological activities including cell proliferation and differentiation 34,35. Recently, another study demonstrated that p38/BDNF coupled signaling mediates neurite outgrowth and neuronal survival 36. Engel et al., have discussed the hypothalamic neurogenesis p38MAPK and BDNF-dependent mechanism 37. A previous study suggested one of the possible downstream pathways of C5a is p38, which showed its role in C5a-induced chemotactic cell migration38.
We also identified the role of p38 in the LPS-induced odontogenic differentiation of DPSCs (manuscript under review). LPS treatment increased the expression of p38, and activated p38, and treatment with the SB20358 abolished the LPS-induced DSPP and DMP-1 increase. These data suggest a crucial role of C5aR and its putative downstream target p38 in the LPS-induced odontogenic DPSCs differentiation. Our results are in accordance that depicts the possible involvement of p38MAPK in C5L2-mediated BDNF secretion.
Taken together, we demonstrated for the first time that C5L2 siRNA silencing enhances the BDNF production in DPSCs, with or without LPS. Our data suggest the negative role of C5L2 and propose the mechanistic aspects of C5L2 mediated BDNF secretion via the p38MAPK pathway (Fig. 6). These data could facilitate the future stem cell therapy direction and use of C5L2 as a target molecule considering nerve regeneration and axonal growth.