In this retrospective, cross-sectional, observational, analytical, case-control study, we evaluated the role of proinflammatory cytokines, particularly IL-10, TNF-α, IL-1, and IL-6, as biomarkers of ischaemic stroke in a large cohort of 429 patients and 195 controls. Our study showed that patients with ischaemic stroke had significantly higher levels of TNF-α, IL-1, and IL-6 than controls, and this finding is consistent with the reported evidence in the literature31,32,33,34. In particular, several authors have shown that patients with ischaemic stroke have higher circulating levels of IL-1 within 24 hours of the acute event compared to controls and patients with other neurological conditions, such as Alzheimer's and Parkinson's disease35,36,37. In contrast, two studies have reported no difference in circulating IL-1 levels between ischaemic stroke patients and healthy controls 12 h and 72 h after symptom onset32,38. These contradictory results between studies can be explained, in part, by differences in the biomarker assessment time or the sample size of the stroke patient cohorts (up to 120 patients for those studies reporting higher levels in stroke patients and less than 50 patients in those reporting no differences).
Similarly, there are conflicting data regarding TNF-α alterations after ischaemic stroke. Notably, some studies have reported that circulating levels of TNF-α do not change after the ischaemic event within the first 24 h32,38. In contrast, others have reported increased blood levels of TNF-α in patients with ischaemic stroke compared to controls39,33,36,40.
However, the lack of differences reported in some studies may be partly due to small sample sizes. In particular, studies that did not report differences included fewer patients (19 to 34 patients with ischaemic stroke) than those showing increased TNF-α levels (23 to 131 patients with ischaemic stroke, including four studies with more than 100 patients). Even concerning IL-10, the evidence in the literature is very heterogeneous. Some authors have described a reduction in IL-10 levels in patients with ischaemic stroke compared to controls, while others have reported no difference between patients and controls41,42. Some authors43 recently showed that patients with ischaemic stroke had significantly increased IL-10 levels compared to controls, in agreement with our results43,44.
The literature seems to agree on the increase in circulating levels of IL-6 in patients with ischaemic stroke compared to controls and patients with other clinical conditions that mimic stroke, such as syncope, seizures, and primary headache disorders45,38,46.
The role of immunoinflammatory activation of the acute phase is further confirmed by our results concerning the analysis of ROC curves, indicating that high serum levels of IL-1beta, IL-6, and TNF-alfa are significantly predictive of stroke diagnosis.
Another interesting finding of our study is the different immunoinflammatory profiles of the various TOAST stroke subtypes. In particular, we reported that the CEI subtype is characterized by a higher inflammatory degree represented by significantly increased TNF-alpha, IL-1, and IL-6 and reduced levels of IL-10 compared to the LAAS and LAC subtypes. This result, in agreement with another study47, further supports the hypothesis that inflammation plays an essential role in the pathogenesis of ischaemic stroke but also suggests the possibility of a peculiar immunoinflammatory pattern for each stroke subtype. The marked inflammatory background that characterizes the CEI subtype agrees with that described in previous reports, indicating the higher severity of this subtype48. Furthermore, strokes of cardioembolic origin are generally more prognostically unfavourable than the other subtypes. This could be associated with an increased production of inflammatory cytokines, which, in turn, could result from an increased recall of inflammatory cells, such as polymorphonuclear cells, as suggested in the study by Licata et al.46.
Finally, the presence of an essential inflammatory component is partly justified by the close association between the CEI subtype and atrial fibrillation (AF). The latter represents the leading cause of cardioembolic stroke. AF is a complex clinical condition with a multifactorial pathogenesis. Numerous pieces of evidence support the role of local and systemic inflammation in the pathogenesis and maintenance of AF49,50. The possible involvement of inflammation in AF derives from the initial observation of a high incidence of this arrhythmia in patients with overt inflammatory conditions of cardiac origin (myocarditis, pericarditis) and noncardiac conditions (pneumonia and inflammatory bowel diseases).
Subclinical inflammatory conditions (for example, in ischaemic heart disease) also contribute to the development of cardiac arrhythmia. Regardless of whether AF is the cause or consequence of an inflammatory process, it is significantly related to oxidative stress associated with myocardial infiltration with inflammatory cells (e.g., macrophages), which is accompanied by the release of reactive oxygen species. Inflammatory conditions lead to activating the renin-angiotensin-aldosterone system (RAAS) and thus to activating nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase. Consequently, these processes trigger the activation of the TGF-β (transforming growth factor-beta) pathway and the structural and electrical remodelling of the myocardium. Thus, increased expression of various inflammatory cytokines and chemokines, such as IL-1, IL-6, and TNF-α, occurs51. Therefore, the close association between AF and cardioembolic stroke could explain the immunoinflammatory phenotype that characterizes cardioembolic stroke.
Given the close association between ischaemic stroke and immunoinflammatory variables, we wondered if there was a relationship between the frequency of some candidate SNPs of genes involved in inflammation and thrombosis/fibrinolysis and the risk of ischaemic stroke. Genetic analysis revealed a significant association between ischaemic stroke and all selected polymorphisms in the genes encoding the proinflammatory cytokines IL-10, TNF-α, IL-1, and IL-6, the clotting factors TPA and PAI-1, and prostaglandin 2. Furthermore, only the rs9509 polymorphism of MMP-9 was significantly associated with ischaemic stroke. Some genotypes were more associated with specific stroke subtypes. Finally, some polymorphisms in the genes encoding prostaglandin 2, MMP-9, and IL-1 were associated with an increased risk of an ischaemic event during follow-up. These results agree with those of other authors who had previously demonstrated an association between some proinflammatory and prothrombotic polymorphisms and the risk of ischaemic stroke16. However, it must be noted that the contribution of individual SNPs to the risk of stroke is small and, therefore, should be evaluated concerning other factors, such as diabetes, hypertension, and dyslipidaemia.
Based on the results obtained relating to the association of cytokines and polymorphisms in ischaemic stroke, we further investigated their possible prognostic role. This analysis revealed that significantly increased levels of TNF-α, IL-1, and IL-6 were associated with a higher incidence of stroke, myocardial infarction (MI), and death during the 8-year follow-up. In comparison, significantly lower levels of IL-10 were associated with a higher incidence of stroke and death. In addition, elevated levels of TNF-α and IL-1 predicted new stroke at five years. These findings are further confirmed by the analysis of Kaplan-Meier curves, strongly indicating how patients with serum levels of IL-1 beta, TNF-alfa, and IL-6 over a specific cut-off showed the highest recurrence rate of new ischemic stroke at follow-up.
Therefore, high levels of cytokines in the acute phase of ischaemic stroke predict the risk of developing another ischemic stroke at follow-up. Our study is the first to demonstrate the importance of inflammatory cytokines as predictive biomarkers in patients with ischaemic stroke. They could be used to stratify patients into various risk classes. Several studies have highlighted an association between altered cytokine levels and worse outcomes, assessed above all in terms of neurological deterioration and disability52. Few studies, on the other hand, have evaluated the association between cytokines and the incidence of events in subsequent years.
To date, a predictive role of cytokines has been described in patients with cardiovascular diseases, such as heart failure and AMI53. In particular, several authors have shown that IL-6 is significantly associated with an increased mortality rate in patients with heart failure54.
Cerebral ischaemia initiates a complex cascade of events at the genomic, molecular, and cellular levels; inflammation at the central nervous system (CNS) and peripheral levels is the protagonist. The full spectrum of inflammatory processes is likely to work in concert. However, cytokines are important mediators of stroke-induced immunological/inflammatory reactions, contributing to stroke progression, disease severity, and outcome. Cytokines can perform various functions during cerebral ischaemia. On the one hand, they can attract leukocytes and stimulate the synthesis of adhesion molecules in leukocytes, endothelial cells, and other cells, thus favouring the inflammatory response of damaged brain tissue; on the other hand, they can facilitate thrombogenesis by inducing an increase in the levels of plasminogen activator inhibitor 1 (PAI-1) and platelet-activating factor and by inhibiting tissue plasminogen activator. Among the cytokines involved in the pathogenesis of ischaemic stroke, TNF-α, IL-6, and IL-1beta seem to play a predominant role.
Several pathogenetic hypotheses could explain the role of cytokines as predictive biomarkers of recurrence of cerebrovascular and cardiovascular events at medium-term follow-up. First, the well-established association between acute-phase cytokine levels and systemic "atherosclerotic burden" (coronary, carotid, vertebrobasilar, and peripheral vascular) could partly explain the predictive role of cytokines. Atherosclerosis is a chronic inflammatory disease, and cytokines are involved in the formation and progression of atherosclerotic plaques. The balance between pro- and anti-inflammatory cytokines is the main factor determining atherosclerotic plaque stability. Therefore, an alteration in cytokine levels characterized by an increase in proinflammatory cytokines and a reduction in anti-inflammatory cytokines can promote instability and the rupture of atherosclerotic plaques, leading to acute events, such as AMI and acute ischaemic stroke8.
A relationship between circulating levels of cytokines and cardiovascular risk factors, such as hyperglycaemia and dyslipidaemia, could be another plausible hypothesis.
Indeed, in our study, we observed a relationship between serum levels of glucose and total and LDL cholesterol in patients with more severe ischaemic stroke. In addition, we found a significant association between all the immunoinflammatory variables and LDL-cholesterol and serum glucose levels. In particular, compared to patients with normal LDL-cholesterol levels, patients with increased LDL-cholesterol levels had significantly increased TNF-α, IL-1, and IL-6 levels and decreased IL-10 levels (Table 7). Similarly, compared to patients with normal blood glucose levels, patients with elevated blood glucose levels had significantly reduced IL-10 levels and elevated TNF-α, IL-1, and IL-6 levels (Table 8). These seem to be original and novel. It has been reported that some proinflammatory cytokines, such as TNF-α, IL-6, and IL-1, cause alterations in the signalling pathways of insulin and lipids, thus influencing insulin sensitivity and lipid metabolism55. In particular, several studies have shown that TNF-α can induce insulin resistance through various mechanisms, such as the downregulation of genes (for example, GLUT-4) indispensable for the biological function of insulin and the induction of an increase in free fatty acid levels by stimulation of lipolysis and negative regulation of peroxisome proliferator-activated receptor-γ (PPAR-γ)56.
It has been shown that in pancreatic β cells, IL-1 can activate the Jun amino-terminal kinase (JNK) pathway, which mediates the suppression of the transcription of the insulin gene57. In addition, IL-1 causes a reduction in the expression of insulin receptor substrate 1 (IRS-1), the inhibition of the translocation of the glucose transporter GLUT-4 to the plasma membrane, and the reduction of the absorption of stimulated glucose insulin and lipogenesis58. Furthermore, in mouse models, it has been observed that IL-1 is able, on the one hand, to promote hepatic steatosis by stimulating the production of triglycerides, the accumulation of cholesterol, and the formation of lipid droplets and, on the other hand, to regulate hepatic insulin resistance and fibrosis59. Conversely, IL-1 inhibition has been found to attenuate steatosis and liver damage60, improve atherosclerosis, and reduce circulating glucose levels61. IL-6 also seems to regulate insulin sensitivity through various mechanisms. This cytokine has been shown to exert long-term inhibitory effects on IRS-1, GLUT-4, and PPAR gene transcription, as well as insulin-stimulated tyrosine phosphorylation and glucose transport, resulting in reduced insulin signalling and action62.
Therefore, a variety of evidence supports the predictive role of cytokines in patients with ischaemic stroke.
Overall, the results of this study further support the importance of cytokines as biomarkers of ischaemic stroke.
The results of the metabolic determinants of immunoinflammatory activation of the acute phase of this disease suggest two possible cross-linking interpretations: the immunoinflammatory activation is the expression of an accentuated dysmetabolic background, or the alteration of metabolic variables is the result of the immunoinflammatory activation during the acute phase of ischemic stroke.
Given the evidence of the critical role of cytokines in both the pathogenesis and progression of ischaemic stroke, some authors have evaluated their possible role as therapeutic targets. This is an area of research that is somewhat controversial since some data obtained from mouse models seem to suggest that the inhibition of inflammatory cytokines increased in the acute phase of ischaemic stroke - by blocking their receptors or using monoclonal antibodies that bind to the cytokine, making it unavailable to its receptor - reduces the volume of the infarcted brain area and thus the outcome of the disease.
A concrete and precise application of this concerns some studies in which the researchers, through mouse models, have evaluated the possibility of inhibiting the nuclear factor kappa-light-chain-enhancer of activated B cells (NFkB) pathway, thereby limiting its effect of promoting transcription of inflammatory cytokine genes: Zeng et al, through tripartite motif containing 9 (TRIM9), a brain-specific ubiquitin ligase, achieved significant inhibition of the NFkB pathway in cell culture in vitro and later found in vivo that systemic administration of a recombinant virus carrying TRIM9 in the brain effectively reduced neuroinflammation and neuron death, especially in older mice with stroke63.
In conclusion, our study highlighted the importance of immunoinflammatory components in the pathogenesis and progression of ischaemic stroke. Moreover, cytokines represent reliable biomarkers clinicians can use to correctly assess and stratify the risk of ischaemic stroke in patients. The genetic analysis highlighted a different genotypic distribution of some of the selected prothrombotic/proinflammatory polymorphisms between patients and controls. However, no association with the prognosis of event recurrence was found. Therefore, further studies are needed to investigate the contribution of genetics to the occurrence of stroke.
Beyond the individual risk related to the expression of polymorphisms of prothrombotic and proinflammatory genes, in addition to medical therapy capable of intervening on metabolic variables, the possibility of affecting the pathogenetic mechanisms of ischemic stroke by minimizing the immunoinflammatory burden represents an optimistic expectation regarding the prognosis and the risk of recurrence of the event and the risk of developing the event itself. The vision that we can modify the inflammatory microenvironment that surrounds and amplifies the ischaemic event and the opportunity to do so upstream of the chain of events that lead to the production of inflammatory cytokines represents a lighthouse in the night for us as researchers because it allows us, on the evidence we have documented, to think about profoundly and decisively changing the natural history of this disease .