Spatio-temporal activation of caspase-8 in myeloid cells upon ischemic stroke
© The Author(s). 2016
Received: 1 July 2016
Accepted: 11 August 2016
Published: 26 August 2016
Ischemic stroke (caused by thrombosis, embolism or vasoconstriction) lead to the recruitment and activation of immune cells including resident microglia and infiltrating peripheral macrophages, which contribute to an inflammatory response involved in regulation of the neuronal damage. We showed earlier that upon pro-inflammatory stimuli, the orderly activation of caspase-8 and caspase-3/7 regulates microglia activation through a protein kinase C-δ dependent pathway. Here, we present in vivo evidence for the activation of caspase-8 and caspase-3 in microglia/macrophages in post-mortem tissue from human ischemic stroke subjects. Indeed, CD68-positive microglia/macrophages in the ischemic peri-infarct area exhibited significant expression of the cleaved and active form of caspase-8 and caspase-3. The temporal and spatial activation of caspase-8 was further investigated in a permanent middle cerebral artery occlusion mouse model of ischemic stroke. Increasing levels of active caspase-8 was found in Iba1-positive cells over time in the peri-infarct area, at 6, 24 and 48 h after artery occlusion. Analysis of post-mortem brain tissue from human subject who suffered two stroke events, referred as recent and old stroke, revealed that expression of cleaved caspase-8 and -3 in CD68-positive cells could only be found in the recent stroke area. Analysis of cleaved caspase-8 and -3 expressions in a panel of human stroke cases arranged upon days-after stroke and age-matched controls suggested that the expression of these caspases correlated with the time of onset of stroke. Collectively, these data illustrate the temporal and spatial activation of caspase-8 and -3 in microglia/macrophages occurring upon ischemic stroke and suggest that the expression of these caspases could be used in neuropathological diagnostic work.
KeywordsMicroglia Macrophage Caspase-8 Caspase-3 Ischemic stroke Human brain tissue pMCAO model Spatio-temporal activation
Every year, an estimate of 15 million people worldwide suffers a stroke. As a result, nearly six million people die and an almost equal number of survivors are left with long-term disabilities [1, 2]. Stroke is an acute cerebrovascular accident, which occur due to deranged blood supply to the brain. There are two main types of stroke: ischemic, due to lack of blood flow, and hemorrhagic, due to bleeding. Ischemic stroke, which is caused by a vessel obstructive thrombosis, embolism or vasoconstriction, accounts for over 80 % of all incidents, is the focus of the present study [1–3].
A decrease or reduction in blood flow results in hypoxia and glucose deprivation, which can lead to neuronal damage and cell death. The center of the ischemic area, the ischemic core, is most affected by the reduction in blood flow and also suffers the more instant and severe damage of the tissue. The area surrounding the ischemic region, the penumbra, can receive low levels of blood flow from adjacent vascularized areas, resulting in slower development of neuronal damage. Injured and dying cells release damage-associated molecular patterns (DAMPs), which activate an immune response that is a major contributor to stroke pathophysiology. In fact, the immune response to acute cerebral ischemia triggers an inflammatory reaction that may last up to several months and plays a critical role in mediating post-ischemic damage of the tissue and secondary neurodegeneration in the penumbra .
The infiltration of blood-borne immune cells facilitated by disruption of the blood–brain barrier integrity following brain ischemic injury contributes to the neuroinflammation process. Nevertheless, the brain’s initial inflammatory response to ischemic event is primarily thought to be mediated by microglia, the brain resident immune cells. Microglia are highly dynamic cells, which constantly scavenge the brain for potential threats and can get rapidly activated upon detection of insults to the brain, danger-signals or changes in the brain microenvironment . In response to the tissue damage, microglia become activated and migrate to the ischemic area. Microglia are a predominant source of proinflammatory mediators including cytokines (e.g. tumor necrosis factor and interleukin-1β), complement factors, free radicals, nitric oxide (NO), chemokines (e.g. CCL2 and CCL3) and prostaglandins, all of which potentially contribute to further neuronal dysfunction and death [6, 7].
Suppression of neuroinflammation using a variety of drugs were proven to be successful in reducing infarct volume and improving outcomes in experimental models of stroke . Despite these promising preclinical trials, up to date, clinical trials using anti-inflammatory agents have failed to improve clinical outcomes . Therefore, in order to revitalize interest for the therapeutic targeting inflammatory pathways for the treatment of acute ischemic stroke there is a need for comprehensive understanding of the time-dependent recruitment and activation of inflammatory immune cells.
Caspases, a family of cysteinyl aspartate-specific proteases, are best known as executioners of apoptotic cell death and their activation are considered as a commitment to cell death. However, caspases also function as regulatory molecules for immunity, cell differentiation and cell-fate determination . We previously reported the existence of a caspase-dependent signalling pathway controlling microglia pro-inflammatory activation and associated neurotoxicity. We showed that the orderly activation of caspases -8 and -3/7, commonly known to have executioner roles in apoptosis, can promote pro-inflammatory activation of microglia in the absence of cell death . Additionally, we recently obtained evidence that the activation of human monocytes/macrophages in response to a pro-inflammatory stimulus also rely on caspase-8 function . In addition, increasing evidence strongly suggests that caspase-8 may play a critical role in IL-1β processing, with special relevance to the NLRP3 inflammasome activation . NLRP3 inflammasome activation usually requires two signals or steps; signal 1 (priming) leading to NF-kB activation and signal 2 (typically ATP or nigericin) leading to NLRP3 machinery assembly (including at least NLRP3, the ASC (PYCARD) adaptor, and caspase-1) . As previously stated, we first demonstrated the involvement of caspase-8 in NF-kB activation (priming) in microglia in response to diverse inflammatory stimuli , recently validated using conditional caspase-8 knockout mice specifically in the myeloid system . The activation of NF-kB is critical for upregulating the transcription of both pro-IL-1β and NLRP3, as both are further needed for inflammasome formation and activation. A role of caspase-8 in mediating priming and activation of the NLRP3 inflammasome has also been demonstrated in primary macrophages . Coimmunoprecipitation and confocal studies have demonstrated that caspase-8 is present in the NLRP3 inflammasome complex, where it is believed to be involved in cleavage and processing of procaspase-1 . It is important to note that IL-1β activates T-cell-mediated innate immunity and promotes secondary ischemic damage during the subacute phase of ischemic brain injury . Given the high number of danger-associated molecular patterns (DAMPs) released as a consequence of the ischemic damage, and the key roles of caspases in regulating brain immune functions, it is important to discern between the apoptotic and non-apoptotic roles of caspases upon ischemic stroke.
In the present study, we investigated in vivo and in post-mortem tissue from ischemic stroke subjects whether caspase-8 and caspase-3 activation, key players of the caspase-dependent signaling pathways regulating microglia and macrophages (MMs) pro-inflammatory activation, exhibited spatiotemporal features upon ischemic stroke.
Materials and methods
Human brain tissue
Human brain tissue from stroke subjects and controls were used in this study as approved by the Regional Ethical Review Board in Lund, Sweden (Dnr 2010-196). Stroke tissue from regions within the white matter of the frontal and parietal lobe was examined and compared to area- and age-matched controls. Both female and male cases were included in the study with an average age of 75 ± 9 years for stroke cases (n = 9) and 75 ± 11 years for controls (n = 5). Deceased individuals referred for autopsy were examined for stroke or/and diffuse ischemic damage in frontoparietal white matter, judged as relevant for particular ischemia susceptibility in the upper border zone areas. Three of these patients died from a clinically suspected or also radiologically ascertained stroke within the last 24 h. In two other patients, an older stroke was known and also detected radiologically in the basal ganglia and the thalamus, however not reported in the white matter, from where the tissue was macroscopically identified and sampled. In four cases found to have ischemic white matter damage, the autopsy referrals focused on cardiopulmonary disease, including myocardial infarcts in some cases. In the latter individuals, macroscopic examination of the cut brain revealed either clear-cut or suspected infarcts, which were later microscopically verified. For sampling from focal stroke, an area of approximately 10 mm2 and 5 mm thickness in the immediate vicinity/border of the lesion was taken for analysis. For diffuse ischemic damage (non-focal lesion, no complete tissue loss) a similar amount of tissue was sampled in the most damaged area.
The tissue was fixed in 4 % formaldehyde solution and embedded in paraffin, in accordance with the standard procedures within the department. Sections of 5 μm thickness were cut with microtome and mounted on glass slides. Tissue sections on regular slides were processed for standard/basic stainings (hematoxylin-eosin (HE) and luxol fast blue/cresyl violet (LFB) for myelin and cell structures. Tissue sections on positively charged glass slides were processed for immunohistochemistry. All brain sections were analyzed under the microscope for clinical diagnostic purpose before inclusion in the study.
Animals and surgery
C57BL/6 mice used in this study were obtained from the Center of Production and Animal Experimentation (Espartinas, Spain). Experiments were performed at the University of Seville (Spain) in accordance with the Guidelines of the European Union Council (86/609/EU), following Spanish regulation for the use of laboratory animals approved by the Scientific Committee of the University of Seville. Animals were kept at diurnal conditions with ad libitum access to food and water. Experiments were conducted using 3-month-old male mice (n = 13). Electrocoagulation was used to induce a permanent middle cerebral artery occlusion (pMCAO), as described elsewhere . Six to 48 h post pMCAO, animals were perfused with 4 % paraformaldehyde, pH 7.4, during deep isoflurane anesthesia and brains were removed and cryoprotected in sucrose. After freezing in isopentane at −40 °C, brains were cut in 25 μm thick coronal sections with a cryostat and mounted onto gelatin coated slides.
Deparaffination of the human tissue sections was done using standard procedures in the department of Neuropathology. The sections were boiled during 15 min in 10 mM Citrate buffer pH 6.0 for antigen retrieval. Stainings were done using an automated immunostainer (TechMateTM 500 Plus, DAKO) with DAKO ChemMate Kit Peroxidase/3-3’diaminobenzidine.
Mouse tissue sections were washed and incubated for 20 min in 0.3 % hydrogen peroxide in methanol. After washes, the sections were incubated in blocking buffer containing 1 % goat serum (Vector) in TBS for 1 h. Incubation with primary antibodies diluted in 1 % goat serum and 0.25 % Triton-X 100 in TBS for 24 h. 3 × 10 min TBS washes was followed by incubation with biotinylated secondary antibodies diluted in 0.25 % Triton-X 100 in TBS for 2 h. Sections were thereafter incubated with ExtrAvidin®-Peroxidase solution (Sigma) and visualized with a standard diaminobenzidine/hydrogen peroxidase reaction for 5 min.
Deparaffination of the human tissue was done using 3 × 5 min incubations in Xylene, followed by 2 × 10 min in 100 % ethanol, 2 × 10 min in 95 % ethanol and 3 × 5 min in H2O.
Antigen retrieval was done for all tissue used for immunofluorescent staining by boiling in 10 mM Citrate buffer pH 6.0 for 10 min at 95 °C. When sections reached RT they were washed in TBS, before blocking in 5 % goat serum and 0.1 % Triton-X100 in TBS for 1 h. Incubation of primary antibody diluted in blocking buffer was done overnight at 4 °C. Washes in 3 × 10 min TBS was followed by 1 h by appropriate secondary antibody in 0.1 % Triton-X100 in TBS in RT. Sections were washed in TBS before 15 min incubation in 1 μg/ml Hoechst 33342 in RT, and followed by TBS wash. Autofluorescence removal reagent (#2160 Millipore) was used for the human tissue according to manufacturer’s recommendations.
Primary antibodies detecting anti-human cleaved caspase-8 (Asp391) (18C8; #9496), anti-mouse cleaved caspase-8 (Asp387) (D5B2; #8592), anti-human and -mouse cleaved caspase-3 (Asp175) (5A1E; #9664), anti-human cleaved PARP (D64E10; #5625) were purchased from Cell Signaling Technology®, anti-human CD68 (PG-M1; #M0876) from Dako and anti-mouse Iba-1 (#NB100-1028) from Novus Biologicals and (#019-19741) from Wako. For immunofluorescence detection, fluorophore conjugated secondary antibodies were purchased from Invitrogen, ThermoFisher Scientific. Goat anti-rabbit Alexa Fluor®488 and goat anti-mouse Alexa Fluor®594 were used for the human tissue analysis, whereas donkey anti-rabbit Alexa Fluor®488 and donkey anti-goat Alexa Fluor®594 were used for mouse tissue analysis. Biotinylated goat anti-rabbit IgG were obtained from Vector Laboratories.
Analysis of human tissue by Immunohistochemistry was done by a neuropathologist with a light microscope and scored for presence of cleaved caspases and upregulation of CD68 in the peri-infarct and ischemic area. Presence of cleaved caspase-8 and -3 as well as increased CD68 was semiquantitative assessed based on staining intensity of each antibody in both peak foci and the entire sampled region. The scoring was done blinded to other clinical information. Additional hematoxylin & eosin (HE) and luxol fast blue/cresyl violet (LFB) staining of all tissue was used to identify the stroke area and to evaluate the age of the ischemic lesions. The same areas were examined for the double immunofluorescent labelling of cells by Zeiss LSM510 and Zeiss LMS700 confocal laser scanning microscopy equipped with inverted Zeiss Axiovert 200 m microscopes using Zeiss LSM 5 and Zeiss ZEN 7.1 software.
Immunohistochemistry cell quantification
Temporal and spatial activation of caspase-8 in Iba1-positive myeloid cells in a pMCAO mouse model of ischemic stroke
To investigate activation of caspase-8 in microglia/macrophages (MMs) upon a stroke event, a mouse model with a permanent middle cerebral artery occlusion (pMCAO) was used to simulate an ischemic stroke. Immunohistochemical labeling of Iba1 was used to detect the MMs present in the ischemic core, peri-infarct area and surrounding area (Fig. 1a). Active caspase-8 was detected using an antibody that recognizes the mouse caspase-8 when proteolytically activated by cleavage at amino acid residue Asp387. Positivity for cleaved caspase-8 was detected in the ischemic core and the peri-infarct area (Fig. 1a) and could be associated with the Iba1-positive cells, as illustrated with immunofluorescence double staining confirming expression of cleaved caspase-8 in Iba1-positive cells upon stroke (Fig. 1b).
Expression of active caspase-8 and caspase-3 in CD68-positive myeloid cells in stroke subjects correlates with age of the ischemic area
Temporal expression of active caspase-8 and caspase-3 in a panel of stroke subjects
Days after stroke
There is compelling evidence that brain injury following ischemic stroke develops from a complex series of pathophysiological events that evolve in time and space [19, 20]. After an ischemic stroke, experimental and clinical data suggest that a prominent inflammatory response develops, propagates, and lasts for many days, and is believed to exacerbate neuronal cell death [21, 22].
The brain’s initial inflammatory response to stroke is proposed to be primarily mediated by microglia, the resident immune cells of the CNS. However, within minutes or hours of the stroke event, the blood–brain barrier is compromised and infiltration of monocytes, neutrophils and lymphocytes occurs [23, 4, 24]. The brain-resident (microglia) and infiltrating peripheral (monocytes) myeloid cells have a prominent role in initiating, sustaining and resolving post-ischemic inflammation. It is therefore of importance to elucidate the molecular mechanism regulating their activation. Our team previously described an unexpected novel function for caspases in the control of microglia activation and thereby neurotoxicity. We showed that orderly activation of caspase-8 and caspase-3 regulates microglia activation, in the absence of cell death . In addition, we recently obtained evidence that caspase-8 regulates the activation of human monocytes . Considering the central role played by these caspases in the activation of microglia/monocytes, and the contribution of these cells in the observed inflammatory response following ischemic stroke, we decided to investigate whether activation of these caspases follow spatial and temporal features.
Immunohistochemical staining, as well as immunofluorescence confocal imaging, of post-mortem tissues from subjects who had suffered an ischemic stroke, was used with a CD68-antibody to detect activated myeloid cells. Additional staining with cleaved caspase-8 or cleaved caspase-3 revealed that myeloid cells in the ischemic core and peri-infarct area expressed active caspase-8 and caspase-3. It is believed that non-apoptotic functions of caspases rely on a moderate activity and a restricted subcellular localization. We have demonstrated that a differential processing of caspase-3 zymogen may ultimately lead to apoptosis (caspase-3 subunit p17; nuclear localization) or microglia activation (caspase-3 subunit p19; cytosolic localization) . Our confocal analysis demonstrated a non-nuclear localization of active caspase-3 within myeloid cells early after stroke, a view that fits well with the non-apoptotic role of caspases in regulating myeloid cell activation. Analysis of brain tissue samples from a pMCAO mouse model of ischemic stroke, at 6, 24 and 48 h post artery occlusion, illustrated a temporal and spatial activation for caspase-8 in Iba1-positive myeloid cells. Indeed, increased levels for cleaved caspase-8 staining were found to correlate with morphological changes of the Iba1-positive cells from ramified cells to amoeboid or rounded shapes in proximity to the ischemic core. Notably, this correlation was particularly evident in the peri-infarct area, a region revealing penumbra like conditions and is potentially salvable upon a brain infarct, in contrast to the stroke core where perfusion is completely absent and irreversible loss of tissue (infarction) occurs within minutes . It has been long established that microglia activation is particularly evident in the penumbra region in response to ischemic damage . Although the contribution of the inflammatory response to ischemic brain injury is under debate, increasing evidence points out a deleterious role. For instance, recent data have demonstrated how microglia in the penumbra region is strongly associated with blood vessels; reactive microglia phagocytose endothelial cells, thus contributing to BBB disruption and neurodegeneration . In the present study we demonstrate the potential contribution of caspases in regulating brain immune function in the peri-infarct penumbra-like region. The multifaceted roles of caspase-8 make this caspase very attractive in the context of future stroke strategies aimed at minimizing brain injury. These varied roles include i) it controls microglia activation through a caspase-3/NF-kB-dependent mechanism, with the subsequent release of neurotoxic proinflammatory cytokines , ii) recent data suggest a prominent role of this caspase in the priming and activation of inflammasomes , iii) inflammasome activation may lead to pyroptosis, a proinflammatory and lytic mode of cell death and iiii) caspase-8 negatively regulates programmed necrosis or necroptosis, which relies of a molecular platform known as necrosome and strongly associated to immune cells of myeloid origin including microglia, monocytes and macrophages . Since the repair process for damaged brain tissues and regeneration of neural cells takes place during resolution of inflammation , and having demonstrated that active caspase-8 is mostly confined to reactive myeloid cells in the peri-infarct region, we anticipate that site-directed delivery of caspase-8 inhibitors under stroke conditions may hinder microglia activation and affect microglia survival. Finally, our analysis of post-mortem tissues from subjects who suffered two independent ischemic stroke events, as well as the examination of panel of 10 different stroke areas of various ages (i.e. time after stroke onset) suggested that the presence of active caspase-8 and -3 in CD68-positive cells correlates with the age of ischemic area.
The approximate time after stroke onset can be determined by neuropathologists and forensic pathologists by the postmortem analysis of ischemic lesions. Parameters used may include analysis of inflammation and myeloid component [29, 30]. The distinct pattern of cleaved caspase-8 surrounding the infarct in our data suggests that immunohistochemical analysis of cleaved caspase-8 could be considered as a relevant additional diagnostic parameter.
Whereas inhibition of caspase-8 and/or caspase-3 could be used as therapeutic strategy to combat the inflammatory response initiated upon ischemic stroke remains controversial. Evidence for neuroprotection have been reported with Quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone (Q-VD-OPh) a third-generation broad spectrum caspase inhibitor , which is able to crosses the blood–brain barrier, in animal models of stroke . However, it remains to be established whether Q-VD-OPh affects microglia/monocytes activation in this disease model and contributes to the neuroprotective effect in that manner. In addition, Q-VD-OPh treatment has been reported to impair the neural stem/progenitor cell response after cortical ischemia in mice, highlighting that caution is warranted using such strategy .
In conclusion, we revealed the temporal and spatial activation of caspase-8 and -3 in microglia/macrophages occurring upon ischemic stroke in human stroke subjects and in a mouse model of ischemic stroke. Overall, the report of a spatio-temporal activation of caspase-8 and -3 in microglia/macrophages occurring upon ischemic stroke indicates that any attempt to target the molecular signaling regulating the detrimental inflammatory response would have to take into account the time window for intervention.
Central nervous system
Danger-associated molecular patterns
Luxol fast blue/cresyl violet
Permanent middle cerebral artery occlusion
We thank the CLICK Imaging Facility supported by the Knut and Alice Wallenberg Foundation.
J.R. is supported by a doctoral fellowship from the Karolinska Institutet Foundations; M.A.B. is supported by a postdoctoral fellowship from Swedish Research Council. This work has been supported by grants from the Swedish Research Council, the Swedish Brain Foundation, the Parkinson foundation in Sweden, the Spanish MINECO/FEDER/UE and the Karolinska Institutet Foundations.
Availability of data and material
JR performed all the experiments except otherwise noted. TD performed the surgery and further dissection of the animal brains. EE did the neuropathology analysis of the individuals with stroke and control cases. AP prepared tissue and participated in the morphological assessment of human brain specimens. MAB, JLV and RMP contributed with in vivo analyses. MAB and EK were involved in study design. JR, JLV and BJ designed the study, analyzed and interpreted the data. JR and BJ wrote the first draft of the manuscript. All authors discussed the results and commented on or edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Used of human brain tissue from stroke subjects and controls was approved by the Regional Ethical Review Board in Lund, Sweden (Dnr2010-196). Animal experiments were performed in accordance with the Guidelines of the European Union Council (86/609/EU), following Spanish regulation for the use of laboratory animals approved by the Scientific Committee of the University of Seville.
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