In
the present study, we studied the possible role of GSDMD-induced
pyroptosis mediated by the AIM2 inflammasome in the pathogenesis of EBI
after SAH. The main findings can be summarised as follows: (1)
Pyroptosis-related proteins mediated by the AIM2 inflammasome were
upregulated in the brain temporal cortex after SAH and in primary
cortical neurons exposed to oxyHb. The expression levels of AIM2, GSDMD,
GSDMD-N, caspase-1, caspase-1 p20 and ASC increased continuously in a
time-dependent manner. (2) The results from the immunohistochemistry and
immunofluorescence staining showed that AIM2 inflammasome-mediated
pyroptosis mainly occurred in brain neurons. (3) The inhibition of AIM2
inflammasome activation by knocking down AIM2 and caspase-1 could
suppress GSDMD-induced neuronal pyroptosis by decreasing the expression
and activation of the pyroptosis-related proteins that are mediated by
the AIM2 inflammasome. (4) Knocking down caspase-1 suppressed not only
GSDMD activation-induced neuronal pyroptosis but also caspase-3
activation-induced neuronal apoptosis. These findings suggested, for the
first time, that pyroptosis could be involved in EBI after SAH through
the AIM2/Caspase-1/GSDMD pathway. Moreover, inhibiting the expression
and activation of caspase-1 could alleviate both pyroptosis and
apoptosis (
Supplementary Figure).
The
pathophysiology of SAH is complicated and involves multiple pathogenic
mechanisms (e.g., inflammation, oxidative stress, and apoptosis). A
growing body of evidence indicates that the inflammatory response plays a
vital role in injury expansion and brain damage after SAH, including
EBI, vasospasm, and delayed neurological deterioration
2. In recent years, inflammasomes have been demonstrated to be important participants in EBI after SAH
10.
The inflammasome is a cytosolic multimeric signalling complex that
responds to invading pathogens and host-derived danger signals such that
its activation leads to caspase-1 activation. Inflammasome activation
proceeds via the formation of a multimolecular complex containing a
receptor, an adaptor such as ASC and the cysteine protease caspase-1
13.
Then, activated caspase-1 triggers the maturation of the
pro-inflammatory cytokines IL-1β and IL-18 and induces pyroptotic cell
death. Several nucleotide-binding oligomerisation domain (NOD)-like
receptors (NLRs), as well as AIM2-like receptors (ALRs), have been shown
to form inflammasomes. Most of the inflammasomes described to date
contain an NLR protein, namely, NOD-like receptor containing pyrin
domain 1(NLRP1), NLRP2, NLRP3, NLRP6, NLRP7, NLRP12, and NLR- and
caspase-activating recruitment domain-containing 4 (NLRC4). However,
only a few inflammasomes have been studied in the CNS, namely, NLRP1,
NLRP2, NLRP3 and AIM2. Among these inflammasomes, the NLRP3 inflammasome
has been the most studied. NLRP3 inflammasome-mediated
neuroinflammation is involved in many acute and chronic CNS diseases,
including SAH
10,14,15.
In SAH, activation of the NLRP3 inflammasome is also essential for the
modulation of pro-inflammatory cytokines, and inhibition of the NLRP3
inflammasome by pharmacological treatment can alleviate brain injury
after SAH
10,16.
In addition to NLRP3, AIM2 is also an important inflammasome involved in CNS infection and injury
17.
In the present study, we collected and analysed CSF samples from
patients with or without SAH. The results from the analysis of
patient-derived CSF samples revealed that the level of the AIM2 protein
in the CSF of the SAH patients was significantly greater than that in
the CSF from non-SAH patients. The higher the Hunt-Hess grade was, the
higher the level of AIM2 that was found in the CSF. In the experimental
SAH model, we also found that the AIM2 inflammasome was activated in
vivo at 24 h post-SAH and that in vitro, the AIM2 inflammasome was
significantly unregulated in cultured cortical neurons 6 h after
incubation with oxyHb and the levels continued to increase for 12 and
72 h. To verify the role of GSDMD-induced pyroptosis mediated by the
AIM2 inflammasome in EBI after SAH, LV was used to knock down the
expression of AIM2. The results showed that AIM2 inflammasome-mediated
pyroptosis was significantly alleviated in EBI following SAH.
As
reported, the AIM2 inflammasome could be activated by aberrant dsDNA,
including bacterial DNA and viral DNA, from pathogens and hosts and by
endogenous self-DNA within the cytosol, damaged DNA within the nucleus,
and self-DNA secreted by exosomes
18.
Wang et al. detected CSF DNA levels in patients with SAH and found that
both nuclear and mitochondrial DNA levels in the CSF were significantly
increased in the patients with SAH compared with volunteers, and the
CSF nuclear and mitochondrial DNA levels were significantly higher on
days 1 and 4
19. Moreover, higher CSF DNA levels were associated with worse outcomes for patients with SAH
19.
This result was consistent with the increased AIM2 in the SAH patients
in our study. Combined with the results of this study showing that the
level of AIM2 in CSF increased significantly within 3 days after SAH, we
found that the elevated level of DNA in the CSF after SAH was
synchronous and consistent with that of AIM2. Thus, we speculate that
pyroptosis mediated by the AIM2 inflammasome is involved in EBI after
SAH. This kind of EBI should be considered, to be exact, a secondary
brain injury.
Recently, some scholars believe that pyroptosis
should be redefined as GSDMD-mediated, rather than caspase-1-mediated,
programmed necrosis
20.
GSDMD was discovered to form a pore and act as an effector for
pyroptosis. In GSDMD-deficient cells, pyroptosis cannot be triggered by
known canonical inflammasome ligands
21.
GSDMD contains ~480 amino acids in two domains, and the N-terminal
gasdermin domain (GSDMD-N) and the C-terminal gasdermin domain (GSDMD-C)
are linked by a long loop. Mounting evidence has demonstrated that
GSDMD plays a key role in CNS injury
22,23.
In our study, we also observed that GSDMD was upregulated after SAH.
Although GSDMD is considered the initiator of pyroptosis, GSDMD-N is the
direct and sole effector of pyroptosis. Activated caspase-1 or
caspase-11 efficiently cleaves GSDMD at a conserved glutamic acid
residue (D276 in mouse and D275 in humans)
21,24.
The cleaved GSDMD unleashes the pro-pyroptotic N-terminal fragment from
the auto-inhibited state maintained by the C-terminus, thus separating
the role of GSDMD into that of GSDMD-N and that of GSDMD-C. The released
GSDMD-N is oligomerized, and only the oligomerised form of GSDMD-N is
able to translocate to the plasma membrane, where it induces cell
rupture and the release of inflammatory cytokines, such as IL-1β
25.
We also observed that the GSDMD-N generated by caspase-1 cleavage forms
an oligomer and migrates to the plasma membrane to kill cells.
Furthermore,
GSDMD-N alone localises to the plasma membrane, as evidenced by GSDMD-N
binding to liposomes in studies in vitro. Binding studies with
liposomes and lipid strips revealed that GSDMD-N has a high affinity for
liposomes containing lipids such as cardiolipin, phosphatidylinositol
4-phosphate [PI(4)P] and phosphatidylinositol 4,5-bisphosphate
[PI(4,5)P] compared with other lipid types
26.
It is through its lipid-binding specificity that GSDMD-N disrupts
plasma membranes, which it perpetrates only when exposed to the
cytosolic PI-containing inner leaflet but not when exposed to the
extracellular outer leaflet, which lacks PI
26.
While GSDMD-N kills from within the cell, its released form does not
harm neighbouring cells. Mounting evidence has shown that the inner ring
diameter of the pore formed by GSDMD-N is estimated to be between 10
and 20 nm
6,27,28. Different methods have revealed a pore consisting of 16 or 24 GSDMD-N units
27,28.
This finding is consistent with the observation we made in the present
study revealing that a large number of pore-like structures appear on
the surface of neurons stimulated by oxyHb. The results of the membrane
protein analysis suggest that the pore-like structure may be caused by
the oligomerization of GSDMD-N at the cell membrane.
In the
present study, we also interfered with the expression of caspase-1 by an
LV. After caspase-1 was knocked down, GSDMD-induced pyroptosis was
markedly reduced in EBI following SAH. Knocking down caspase-1 inhibited
not only the expression and activation of GSDMD but also the expression
of pyroptosis-related proteins mediated by the AIM2 inflammasome. We
propose two explanations for this result: First, caspase-1-mediated
inflammatory injury could be greatly alleviated after caspase-1 is
knocked down. Caspase-1 is unequivocally required for the proteolytic
processing of IL-1β and IL-18; these two cytokines engage their
transmembrane receptors IL-1R and IL-18R to promote inflammation via the
activation of nuclear factor-kappaB transcriptional programmes that are
induced by the activation of myeloid differential protein-88, a key
signalling adaptor
29.
When caspase-1 was knocked down, caspase-1-mediated inflammatory injury
may have been alleviated, and the amount of dsDNA released from damaged
cells could be reduced. Second, the non-functional AIM2/ASC complex was
quickly depleted. Juruj et al. found that a negative feedback loop
controlled by ASC/caspase-1 regulates AIM2 complex formation/stability
13.
In the absence of a functional AIM2 inflammasome, the AIM2/ASC complex
formed very rapidly. However, this complex was then removed through
activated autophagy following AIM2 speck formation
13.
Thus, when caspase-1 levels were deficient, the AIM2 inflammasome was
not readily formed and pyroptosis was thus mediated after SAH.
Intriguingly,
knocking down caspase-1 alleviated not only GSDMD activation-induced
neuronal pyroptosis but also caspase-3 activation-induced neuronal
apoptosis. A few other studies have reported that GSDMD-deficient cells
may die because of the caspase-1 cleavage of caspase-3/7
21,30. Inhibiting caspase-1 activity may have attenuated caspase-3-dependent apoptosis, but the mechanism remains unclear
31,32,33.
We found that caspase-1 was essential for the activation of caspase-3.
The cleavage of caspase-3 was significantly hindered when caspase-1 was
knocked down in EBI following SAH. However, the expression of caspase-3
was not affected by caspase-1 knockdown. Therefore, we speculated that
apoptosis was also alleviated in EBI after SAH, because knocking down
caspase-1 hindered the cleavage of caspase-3. It was recently reported
that GSDMD was also cleaved during apoptosis. GSDMD was cleaved by
caspase-3/7 at D87 during apoptosis, while GSDMD was cleaved by
caspase-1 at D275/D276 during pyroptosis
34.
The p30 N-terminal fragment of GSDMD (GSDMD p30) released by the
cleavage at D275/D276 forms pores in the plasma membrane, thereby
mediating pyroptotic cell death. Nevertheless, this cleaved GSDMD also
generated the p43 fragment of GSDMD (GSDMD p43) at D87 during apoptosis,
which inactivated or failed to trigger pyroptosis (
Supplementary Figure).
We preliminarily found that caspase-1 may be an important protein at
the intersection of the pyroptosis and the apoptosis pathways.
Inhibition of caspase-1 activity alleviated not only GSDMD
activation-induced neuronal pyroptosis but also caspase-3
activation-induced neuronal apoptosis.
In this study, we
demonstrated, for the first time, that the GSDMD-induced pyroptosis
mediated by the AIM2 inflammasome may be involved in EBI after SAH. When
inhibiting the activation of the AIM2 inflammasome by knocking down
AIM2 and caspase-1 with LV, the pyroptosis mediated by the AIM2
inflammasome was significantly alleviated. The activation of caspase-3
was also decreased after caspase-1 was knocked down. However, a clearer
regulatory mechanism between pyroptosis and apoptosis remains to be
further explored.
