4 Discussion
Neuroinflammation is a key feature of the aging process, neurodegenerative diseases and injuries that affect the CNS, and is characterized by the activation of microglia and astrocytes. These cells have a fundamental role in the regulation of neuroinflammation, depending on the nature of their activation, they can lead to the production of pro and / or anti-inflammatory mediators and have both beneficial and detrimental effects on neurons (Kotas & Medzhitov, 2015; Medzhitov, 2008). In this work, we sought to evaluate the role of α-Klotho in the modulation of inflammatory processes induced by LPS in glial cells. It is well known that activation by LPS in those cells leads to increased production and secretion of pro-inflammatory cytokines (including TNF-α, IL-6, IL-1, IL-12). In addition, LPS increases expression of other pro-inflammatory mediators, such as chemokines (like CXCL8, CCL5, and CCL2), complement system proteins (like C3, C3aR, C5a5, and factor B) and enzymes (like cyclooxygenase type 2 (COX-2) and induced nitric oxide synthase (iNOS) (Nazem, Sankowski et al., 2015; Rivest, 2009). Our data confirmed these data since LPS increased the secretion of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-1β, and IFN-γ.
Pro-inflammatory mediators, such as TNF-α, IL-1β, and IL-6, when produced in excess and/or in a chronic manner by glial cells can lead to neuronal death (Allan & Rothwell, 2001; Heneka, Kummer et al., 2014). Studies have already shown that the application of GCM of microglia and mixed glial culture activated by LPS in neurons leads to neuronal death (Dai, Yuan et al., 2019; Guadagno, Xu et al., 2013; Sun, Shen et al., 2018; Yshii, Denadai-Souza et al., 2015). According to these data, our data showed that the application of GCM from LPS-activated GCM in neurons led to a dose-concentration dependent increase in neuronal death. As the GCM was used, it is not possible to say which molecule is leading to neuronal death. Probably not just one, but a set of mediators, including the proinflammatory cytokines that were elevated after LPS stimulation such as TNF-α, as we previously demonstrated (Yshii, Denadai-Souza et al., 2015).
The activation of glial cells by LPS leads to an increase in the production of proinflammatory mediators in glial cells and these mediators are capable of inducing neuronal death (D’Angelo, Astarita et al., 2017). Therefore, we investigated whether α-Klotho would be able to decrease the effects induced by LPS in glial cells, since protective and anti-inflammatory activity of α-Klotho protein has already been seen in the renal, vascular and pulmonary systems (Kuro-o, 2019). However, the protective effect of α-Klotho protein in neuroinflammation has been poorly studied. α-Klotho has been shown to decrease NF-κB activation and reduce the production of pro-inflammatory cytokines, such as TNF-α, IL-6, IL-8, and IL-1β, in vivo and in vitro in models of cardiac inflammation (Guo, Zhuang et al., 2018; Hui, Zhai et al., 2017), kidney disease (Zhao, Banerjee et al., 2011) and lung disease (Krick, Baumlin et al., 2017; Li, Wang et al., 2015). For example, in human kidney embryonic cells (HEK293), Zhao et al. demonstrated that pretreatment with 200 pM of α-Klotho for 45 minutes was able to decrease NF-κB activation by approximately 70% and reduce the expression of IL-8, MCP-1, RANTES, and IL-6 after the addition of TNF-α (Zhao, Banerjee et al., 2011). In HUVECs cells, the same pretreatment for 6 hours has been shown to decrease NF-κB activation and expression of adhesion molecules, intercellular adhesion molecule 1 (ICAM-1) and adhesion molecule of vascular cell 1 (VCAM-1), induced by TNF-α (Maekawa, Ishikawa et al., 2009).
Recent studies have demonstrated the protective and anti-inflammatory effect of α-Klotho in the CNS. α-Klotho overexpression in the mouse choroid plexus improved behavioral deficit and increased the number of live neurons after cerebral hypoperfusion, accompanied by a decrease in translocation p65 from the cytoplasm to the nucleus, production of proinflammatory cytokines and activation of astrocytes and microglia (Zhou, Li et al., 2017). A recently published study showed that α-Klotho’s systemic overexpression in an experimental model of amyotrophic sclerosis (mouse transgenic for superoxide dismutase 1), led to later onset and progression of the disease and increased survival of these animals (Zeldich, Chen et al., 2019). Still, it was observed that α-Klotho decreased the expression of inflammatory markers and prevented neuronal death. In addition, a reduction in the secretion of TNF-α, IL-6, and the expression of iNOS and COX-2 induced by LPS / IFN-γ in mouse microglia culture that overexpressed α-Klotho (Zeldich, Chen et al., 2019).
In our study, α-Klotho was able to decrease the secretion of pro-inflammatory cytokines, TNF-α and IL-6. Pretreatment with 1 nM α-Klotho for 4 and 24 hours and 2 nM for 1, 4, and 24 hours decreased LPS-induced TNF-α secretion in glial cells. Also, pretreatment with 1 nM α-Klotho for 24 hours reversed the increase in IL-6 secretion induced by LPS.
Interestingly, experiments in astrocytes purified glial cells reforce the anti-inflammatory action of α-Klotho as pretreatment with this recombinant protein significantly reduce nuclear translocation of Rel A (p65) subunit of NF-κB induced by LPS as revealed by immunofluorescence and Westering blotting experiments. In addition, EMSA data confirmed that α-Klotho was able to revert the p65/p50 NF-κB activation induced by LPS suggesting a putative neuroprotective action of this protein. The present data reforces the importance of understanding the roles of astrocytes and microglia in the neurodegenerative diseases to develop effective therapies to neurodegenerative diseases (Kwon & Koh, 2020). Considering that insulin and glutamate -induced neuronal secreted α-Klotho plays a important role in brain metabolism and neuroinflammation by modulating neuronastrocyte coupling (Mazucanti, Kawamoto et al., 2019) it would be important to consider it an important player in the complex activated glial cells during degenerative diseases.
In fact, when the conditioned medium of glial cells was preincubated with α-Klotho (1 nM) for 24 hours before they being challenged with 1µg/mL LPS for 8 hours (GCM-KL), the neuronal death caused by the GCM of glial cells treated only with LPS was rescued in a lower concentration of GCM (25%), reinforcing, the therapeutic potential of α-Klotho in the CNS shown in other studies.
In fact, it is unclear which mediators were leading to neuronal death as well as to the neuroprotective effect. It may have been due to the decrease in TNF-α and IL-6 levels, which were observed in this study, as well as the decrease in other inflammatory mediators not evaluated or by inducing an adaptive response linked to an increase in protective factors, such as BDNF, or GNFT. In addition, α-Klotho may have had this protective effect due to the secretion of anti-inflammatory mediators and /or neuroprotective factors. Studies that have demonstrated an anti-inflammatory effect of α-Klotho observed a decrease in NF-κB activation (Maekawa, Ishikawa et al., 2009; Zhao, Banerjee et al., 2011). This modulation of NF-κB activation was also observed in the CNS (Zhou, Li et al., 2017). Our data confirmed that at least part of the mechanism by which α-Klotho could be leading to this anti-inflammatory and neuroprotective effect is mediated by NF-κB. The pretreatment with α-Klotho led to a decrease in the activation of NF-κB in astrocytes and, consequently, resulted in a decrease in the production and secretion of proinflammatory cytokines. It is interesting that previous studies from our laboratory showed that α-Klotho has a strong influence in the astrocytic metabolis stimulating aerobic glycoliysis and lactate release mediated by FGFR1 and Erk1/2 activation (Mazucanti, Kawamoto et al., 2019). The ability of α-Klotho to modulate the FGFR and NF-κB signaling in astrocytes is consistent with results in other cell types (Wang, Liu et al., 2019) and suggest that in CNS α-Klotho can modulating neuroinflammation by neuron-astrocyte coupling action.