In the present study, we found a regulatory role of LLLT for microglial functions. Our major findings showed that LLLT significantly reduced LPS-activated microglia-induced neuronal cell death. In LPS-activated microglia-like BV-2 cells, LLLT attenuated inflammation cytokine TNF-α, decreased the production of NO, down-regulated overexpression of iNOS, and caused MyD88 degradation. Another important observation was that microglial phagocytosis was improved after LLLT treatment, characterized by an increase in Rac1 activity, actin polymerization and the ability of the microbeads to phagocytize. Moreover, we found that LLLT could induce the enhancement of microglial phagocytic activity by a Src-dependent PI3K/Akt pathway. Understanding the mechanism and functional significance of LLLT-induced microglia-mediated phagocytosis and neuroinflammation may lead to new neurotherapies.
AD is known to be associated with neuroinflammation and activated microglia. Uncontrolled and excessive activation of microglia is capable of releasing various potentially cytotoxic molecules such as NO, oxygen radicals, and proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 [37, 38]. Chronic neuroinflammation induced by excessive production of these neurotoxic molecules plays an important role in the degenerative process of AD patients. LPS acts as a potent stimulator of microglia and has been used to study the inflammatory process in the pathogenesis of AD and anti-inflammatory therapy for AD treatment. Evidence from some rat models showed that microglia were activated immediately after LPS injection. Significant elevations of cluster differentiation marker CD45, glial fibrillary acidic protein (GFAP), scavenger receptor A (SRA), and Fcγ receptor mRNA were seen after 24 h . LPS-induced inflammation also exacerbates phospho-tau pathology in rTg4510 mice .
TLRs play a key role in microglia-mediated neuroinflammation. Previous studies revealed that integrin CD11b could be activated by TLR-triggered inside-out signaling, and then negatively regulated TLR-induced inflammatory response by promoting degradation of their adaptors . Similar to the results of the present study, Mallard et al. demonstrated that microglial MyD88 signaling played an important role in regulating acute neuronal toxicity and MyD88 deficiency attenuated release of microglial proinflammatory cytokines following LPS exposure .
Much attention has been paid to therapeutic strategies aimed at controlling microglia-mediated neurotoxicity. LLLT is a non-thermal irradiation using light in visible to near infrared range which has been used clinically to accelerate wound healing and reduce pain and inflammation in a variety of pathologies [42, 43]. Moreover, transcranial LLLT has shown good effects on treatment of stroke, traumatic brain injury, and neurodegenerative disease . Although in many pre-clinical and clinical studies the 810-nm NIR light has been used for nerve repairs [45–47], the effects of laser irradiation with different wavelengths on microglial activation remain unclear. Studies had shown that the 632.8-nm laser had advantages over other wavelengths in treating neurological diseases [48, 49]. Therefore, LLLT using the 632.8-nm laser may have high clinical relevance. Recently, it has been debated whether He-Ne laser light can activate a number of signaling pathways including MAPK/ERK, Src, Akt and RTK/PKCs signaling pathway [11, 21, 24, 50].
We explored the role of Src activation involved in LPS-activated microglia after LLLT treatment. In our experiments, we demonstrated that LLLT triggered a significant activation of Src in LPS-activated BV-2 cells (Figure 1C). In addition, the activation of Syk triggered by LLLT was Src-dependent. LLLT-mediated Src/Syk activation could significantly decrease MyD88 and iNOS expression (Figure 2B). Blockade of Src activation or knockdown of Syk negatively affects neuronal survival under LLLT treatment (Figure 2A).
Excessive accumulation of NO has long been known to be toxic to neurons . Oxygen-free radicals such as superoxide can react with NO to form deadly intermediates such as peroxynitrite. Our results indicated that LLLT could efficiently reduce LPS-activated microglia-induced iNOS (Figures 1D and 2C) by downregulating TLR-triggered proinflammation (Figure 2D - I).
How does LLLT activate Src? One of the most plausible explanations is that LLLT activates Src through reactive oxygen species (ROS). LLLT has been demonstrated to increase the level of intracellular ROS generation . With LLLT treatment, light is absorbed by endogenous photosensitizers (porphyrins or cytochromes) that dominantly locate at plasma membrane, mitochondria or lysomes. The photosensitizers’ activation results in ROS (1O2, O2
-, and H2O2) production . Intracellular oxidants could mediate the activation of Src . This hypothesis was also supported by our previous work . Although there may be many other contributors responsible for LLLT-mediated neuroprotective effect, our experimental results suggest that Src and Syk are primary participants in downregulation of TLRs-triggered neuroinflammatory signaling pathway (Figures 2 and 3).
The deposition of Aβ in the extracellular space of the brain plays an important role in microglial activation in AD. Although the role of microglia-mediated inflammation in the pathogenesis of AD is obvious, microglia have been reported to mediate the clearance of Aβ through receptor-mediated phagocytosis, which could delay the progression of AD.
Recent studies suggested that Aβ oligomers could induce a potent inflammatory response and subsequently disturb microglial phagocytosis and clearance of Aβ fibrils, thereby contributing to an initial neurodegenerative characteristic of AD . To address whether LLLT-mediated anti-inflammatory effects can improve microglial phagocytosis, we investigated the microglial phagocytic activity after LLLT treatment. Phagocytic activity of the LPS-activated microglia was markedly enhanced after LLLT treatment (Figures 3 and 6). Furthermore, LLLT could also activate the PI3K/Akt signal pathway (Figure 5), which was dependent on Src activation under LLLT treatment. Since phagocytosis is a Rac1-mediated actin-based process, our results demonstrated that not only the activity of Rac1 but also the F-actin accumulation were increased by PI3K/Akt after LLLT. A constitutively active form of Rac1 greatly increased F-actin polymerization, while a dominant negative form of Rac1 inhibited F-actin polymerization under LLLT treatment (Figure 5B). Thus, these results suggested that LLLT-induced Src activation could also improve microglial phagocytic activity by PI3K/Akt/Rac1 signal pathway.
Activation of the PI3K/Akt signaling pathway has been correlated with tumor metastasis and invasion . Indeed, PI3K is a key signaling molecular for integrin activation and regulation of actin reorganization . The nonreceptor tyrosine kinase FAK-Src complex can initiate a cascade of phosphorylation events to trigger multiple intracellular pathways, including MAPK/ERK and PI3K/Akt signaling [57, 58]. In this study, using LPS-activated BV-2 cells, we found that LLLT-mediated anti-inflammatory effect did not require FAK. One of the most plausible explanations of the above results is that Syk activation does not require actin polymerization since it is unaffected by inhibitors such as cytochalasin D, whereas FAK activation requires actin polymerization [59, 60]. Thus, activation of Syk, but not FAK, plays a key role in Src-mediated anti-inflammatory signal under LLLT. However, inhibition of FAK by transfecting BV-2 cells with FRNK, a naturally occurring inhibitor of FAK signaling, contributed to partial inhibition of microglia phagocytosis.
FAK translocation between cytosolic and membrane is highly regulated by many factors, including tyrosine phosphorylation and actin assembly . Since p85 was associated with FAK to further activate Akt by binding to tyrosine phosphorylated residue 397 of FAK , this may explain why FAK could also be activated before actin polymerization and only had partial effects on Src-mediated microglial phagocytosis. Our results suggest that LLLT-induced phagocytic activity depends on Src-mediated PI3K/Akt signaling pathway, partially due to the phosphorylation of FAK.
Microglial activation is considered as a hallmark of AD. Alternatively, microglial activation is usually associated with marked increase in CD11b expression . Integrin CD11b/CD18 (macrophage antigen complex 1, MAC1, also known as complement receptor 3, CR3) is essential for the phagocytosis of multiple compounds and mediates the activation of phagocytes in response to a diverse set of stimuli . The MAC1 receptor is located on microglia, suggesting its role in neurodegeneration. In addition, previous reports indicated that MAC1 might be a key receptor for Aβ to activate microglia to produce extracellular superoxide, resulting in neurotoxicity . Conversely, others indicated that ROS was a key player in microglial activation in which ROS increased microglial expression of CD11b via NO . In fact, a recent study showed that activation of CD11b via inside-out signaling negatively regulated TLR-triggered proinflammatory response .
It is important to note that the role of microglial activation in AD is still debated, and a simplistic view of microglia as solely beneficial or detrimental cells does not reflect the complexity of microglial function . Functionally, microglia react in diverse ways: they secrete inflammatory mediators, proteolytic enzymes or neurotrophic factors, and are also able to take up soluble and insoluble molecules. Hence, the ideal microglia-targeted AD treatment modalities should not only focus on microglial neurotoxic characteristics, but also on the phagocytic activity.
However, in this study, we used human neuroblastoma SH-SY5Y as the target neuronal cells to mimic responses of inflammation-mediated neurotoxicity. Given that microglia may have recognized markers of malignancy, our results need to be further confirmed using primary cortical neurons in the future studies.
Taken together, the current investigation demonstrates that LLLT can inhibit LPS-activated microglia-induced neurotoxicity and enhance its phagocytic activity through activation of non-receptor tyrosine kinase Src. Although cultured mouse microglia and its treatment with etiological reagents may not truly resemble microglia in the brain of patients, our results suggest that targeting Src may be an important step for the attenuation of microglial activation. Better understanding of the regulation mechanism of activated microglia may provide a therapeutic strategy to control the progression of neurodegenerative diseases.