The development of ischemic brain damage is dramatically affected by the immune system,
whose activation occurs immediately after the insult and may last for several days,
involving a complex interplay between soluble and cellular mediators (Amantea et al.,
2015). Accordingly, recent expression profiling studies have revealed that the majority
of the genes modulated in the blood of stroke patients participate in the regulation
of innate immune responses (Brooks et al., 2014). Moreover, in the clinical setting,
serum levels of markers of acute inflammation correlate with the severity of ischemic
brain damage and neurological deficit.
Both local and systemic inflammatory responses, involving soluble messengers and specialized
cells activated in the brain or recruited from the periphery, exert a dualistic role
on the development of ischemic cerebral injury. Brain resident microglia and blood-borne
immune cells crucially contribute to the acute and chronic processes implicated in
tissue injury, as well as to the regenerative and reparative mechanisms that limit
the damage and provide tissue healing and recovery. In this context, an attractive
approach to improve successful clinical translation of stroke therapeutics would consist
in achieving a rational modulation of the immune system, by blocking its detrimental
inflammatory responses while promoting its beneficial components. This perspective
commentary will focus on the most recent findings regarding relevant targets and drugs
for immunomodulation in stroke and their potential application in patients.
The reduction of cerebral blood flow caused by the ischemic insult prompts rapid neuronal
death in the ischemic core regions and triggers the release of adenosine triphosphate
(ATP) and danger associated molecular patterns (DAMPs) that stimulate purinergic and
specific pattern recognition receptors (e.g., toll-like receptors), respectively,
causing activation of astrocytes and microglia. Depending on the specific phenotype
triggered by the environmental stimuli, microglia may be prompted to release inflammatory
molecules, such as interleukin (IL)-1 and tumor necrosis factor (TNF), or to acquire
an amoeboid morphology endowed with phagocytic activity that clears the damage and
promotes repair. Moreover, activated microglia, damaged neurons and virtually all
the other components of the neurovascular unit release toxic mediators, including
cytokines, proteases and free radicals that prompt blood-brain barrier rupture and
brain infiltration of circulating leukocytes. Thus, signals generated from the brain
are implicated in the peripheral activation and in the cerebral recruitment of neutrophils,
monocytes and lymphocytes that actively participate in the detrimental inflammatory
processes that contribute to ischemic tissue damage, as well as in the beneficial
and immunoregulatory mechanisms that provide tissue repair. In this context, cerebral
ischemia is no longer considered a pathology that affects solely the brain, but represents
a complex disease in which the neuro-immune crosstalk plays a crucial role. This underscores
that, beyond neuronal mechanisms of toxicity, understanding the spatio-temporal evolution
of the recruitment of distinct immune cells and their polarization towards specific
subtypes is crucial to define their role in ischemic brain damage and to identify
novel targets for an effective immunomodulation.
Notably, the majority of the genes acutely regulated in the blood of stroke patients
are expressed in neutrophils and, to a lesser extent, in macrophages (Brooks et al.,
2014). Neutrophils are the first blood-borne cells to be recruited in the ischemic
brain and, although some studies have argued that they are unable to penetrate the
brain parenchyma, their pivotal role in the pathogenesis of ischemic brain damage
is clearly established. In patients, higher peripheral leukocyte and neutrophil counts,
but not lymphocyte counts, are associated with larger infarct volumes, and brain accumulation
of neutrophils correlates with poor neurological outcome and brain damage severity
both in humans and in rodents. Indeed, neutrophils exert detrimental effects by microvessel
obstruction/thrombosis and by releasing a plethora of cytotoxic molecules, including
reactive oxygen species (ROS), reactive nitrogen species (particularly peroxynitrite)
and proteases (such as matrix metalloproteinase-9, MMP-9) endowed with injurious effects,
as demonstrated by the evidence that neutrophils depletion, as well as blockade of
their trafficking, is neuroprotective in focal stroke. Nevertheless, neutrophils also
possess the ability to polarize towards beneficial N2 phenotypes (
Figure 1
). Accordingly, in a recent study, Cuartero et al. (2013) have originally demonstrated
that activation of the nuclear peroxisome proliferator-activated receptor (PPAR)-γ
prompts neutrophil reprogramming toward the N2 phenotype in the setting of stroke
(Cuartero et al., 2013). Thus, the PPAR-γ agonist rosiglitazone exerts neuroprotection
and promotes resolution of inflammation by triggering N2-polarization and increased
neutrophil clearance after experimental stroke induced by permanent middle cerebral
artery occlusion (MCAo) in rodents (Cuartero et al., 2013). Although the exact mechanism
by which N2 neutrophils rescue the ischemic brain is not clear yet, these findings
suggest that the use of N2-polarizing drugs may be beneficial for stroke therapy.
Figure 1
Polarization of innate immune cells and their function in stroke.
Various stimuli, including cytokines and certain receptor ligands, promote polarization
of microglia/macrophages and neutrophils towards specific phenotypes. Based on their
ability to release pro-inflammatory and detrimental mediators (M1 or N1 phenotypes)
or immunomodulatory and pro-survival factors (M2 or N2 phenotypes), innate immune
cells participate in the development of ischemic tissue damage or provide tissue healing
and recovery especially during the late phases of the disease. BDNF: Brain-derived
neurotrophic factor; CKLF-1: chemokine-like factor 1; IGF: insulin-like growth factor;
IL: interleukin; IL-1ra: interleukin-1 receptor antagonist; INF: interferon; i-NOS:
inducible nitric oxide synthase; MIP: macrophage inflammatory protein; NGF: nerve
growth factor; NO: nitric oxide; PPAR: peroxisome proliferator-activated receptor;
RNS: reactive nitrogen species; ROS: reactive oxygen species; RXR: retinoid X receptors;
TGF: transforming growth factor; TLR: toll-like receptors; TNF: tumor necrosis factor;
VEGF: vascular endothelial growth factor.
At variance with the studies focussing on neutrophils, polarization of microglia/macrophages
has been more extensively investigated (
Figure 1
). Their switch towards a multitude of phenotypes is strongly dependent on specific
microenvironmental signals related to the spatio-temporal progression of ischemic
brain damage (Fumagalli et al., 2015). Early after an ischemic insult, local microglia
and newly recruited macrophages assume the M2 “beneficial” phenotype, to then develop
into a pro-inflammatory M1 phenotype upon priming by ischemic neurons. Microglial
cells then shift from a ramified to an ameboid macrophage-like shape, associated to
phagocytosis and to the release of inflammatory mediators (Amantea et al., 2010).
At late differentiation state, microglia assumes a phenotype indistinguishable from
blood-borne macrophages, bearing similar antigenic and morphological features (Fumagalli
et al., 2015). Although phagocytosis clears cell debris and contributes to the resolution
of inflammation, microglia have also been shown to engulf salvageable neurons thus
increasing brain damage.
Likewise microglia, macrophages infiltrating the ischemic brain may exert a dualistic
role on the development of tissue damage depending on their polarization status (
Figure 1
). The M1 phenotype initiates and sustains inflammation by releasing neurotoxic factors
(i.e., TNF-α, IL-1β, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory
protein (MIP)-1α, and IL-6) and ROS that underlie macrophage/microglia-mediated neurotoxicity
after stroke; whereas, M2-polarized cells are involved in beneficial responses by
clearing debris and by promoting angiogenesis, tissue remodeling and repair.
The dualistic role exerted by the cellular mediators of the innate immune reaction
may explain why most anti-inflammatory approaches, conceived disregarding the potential
beneficial function of the target, have failed to reach the clinical setting (Amantea
et al., 2015). In this context, an attractive opportunity to develop novel effective
stroke therapeutics consists in reducing the detrimental responses, while promoting
the endogenous neuroprotective reactions of the innate immune system. Recently, this
concept has been validated in the preclinical setting by studies demonstrating the
neuroprotective effects of shifting the polarization of immune cells towards non-inflammatory
phenotypes in stroke animal models.
Endogenous production of the M2-polarizing cytokine IL-4, triggered by MCAo in mice,
promotes Th2 polarization and, thus, beneficial effects on stroke outcome (Xiong et
al., 2011). Moreover, Gliem et al. (2012) have reported that a subpopulation of bone
marrow-derived monocytes/macrophages, recruited via CCR2 and acting through transforming
growth factor (TGF)-β1, preserves the integrity of the neurovascular unit in murine
stroke models. A decreased expression of M1 markers, together with a partial preservation
of the ischemia-induced expression of M2 markers has been suggested to underlie the
amelioration of stroke outcome observed in myeloid-specific mineralcorticoid receptor
knockout mice (Frieler et al., 2011). Similarly, deficiency of the fractalkine receptor
CX3CR1 triggers a protective inflammatory milieu, characterized by the elevation of
markers of M2 polarization (Fumagalli et al., 2015). Despite this little evidence,
the molecular processes that regulate macrophage polarization in stroke have not been
completely clarified, and further studies are needed to understand whether the acquisition
of a specific phenotype stems from recruitment of circulating precursors or in situ
cell re-instruction.
Moreover, to date, only few experimental studies have evaluated the therapeutic benefits
of drugs acting by increasing the M2/M1 ratio in stroke. Among these, minocycline
has been reported to promote neurovascular remodeling during stroke recovery by facilitating
alternative activation of microglia/macrophages towards a non-inflammatory protective
phenotype (Yang et al., 2015). By following the concept of drug repurposing, we have
recently demonstrated that acute treatment with the macrolide antibiotic azithromycin
attenuates blood-brain barrier damage and cerebral ischemic damage in rodents subjected
to MCAo, with a significant amelioration of neurological deficits up to 7 days after
the insult (Amantea et al., 2016). Up-regulation of M2 markers has also been shown
to underlie neuroprotection by Exendin-4, a glucagon-like receptor 1 agonist clinically
used against type 2 diabetes in young healthy and in aged diabetic/obese mice subjected
to middle cerebral artery occlusion (Darsalia et al., 2014). Interestingly, another
drug widely used for the treatment of type 2 diabetes, metformin, has shown promising
results in stroke animal models based on its immunomodulatory properties. Metformin
is well-recognized as an activator of adenosine 5’-monophosphate-activated protein
kinase (AMPK). In mice subjected to MCAo, chronic metformin treatment promotes functional
recovery and tissue repair via AMPK-dependent skewing of microglia/macrophages toward
an M2 phenotype (Jin et al., 2014).
Thus, the preclinical findings highlighting the neuroprotective potential of M2- or
N2-polarizing agents in stroke are increasing, although further studies are needed
to better investigate the molecular targets that mediate immune cell shift towards
beneficial phenotypes. In order to add significance to such findings, the relevance
of M1-to-M2 or N1-to-N2 polarization for stroke outcome should also be validated in
the clinical setting. In fact, although ischemic stroke is a leading cause of mortality
and long-term disability worldwide, the only treatments available to date consist
in blood flow restoration by lysis or removal of the thrombus. This, together with
the fact that these procedures can be applied to less that 10% of patients due to
their narrow therapeutic window, highlights the urgent need of more effective and
safe stroke therapeutics. In this context, it is interesting to note that the majority
of the studies discussed herein, concerning effective immunomodulatory compounds in
stroke models, are based on repurposing existing drugs characterised by a well established
safety profile in human. This drug discovery approach allows to dramatically reduce
the risk of clinical failure due to undesired side effects or unsuccessful validation
of the target, and is therefore a promising strategy to implement the clinical translation
of immunomodulatory therapies for stroke.
The author thanks all the collaborators who contributed to the research papers upon
which the present commentary is based. Due to space limitations, the author regrets
the omission of many important studies and their corresponding references.