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.