Circulating immunocytes and complements are correlated with severity of ischemic stroke

Immune factors are found to be involved in the pathophysiological process of ischemic stroke. However, the clinical role of the immune system in stroke remains unclear. Our study was designed to investigate the correlation between immunocytes (T cell, B cell, NK cell), complements and stroke severity. Methods 236 patients with rst-ever ischemic stroke were included in our study and divided to mild, moderate and severe groups according to NHISS score at stroke onset. Modied Rankin Scale (mRS) was used to assess short-term prognosis 3 months after stroke. We also collected clinical data and test circulating T cells, CD8 + T cells, CD4 + T cells, B cells, NK cells, C3, C4, complement factor B (CFB) of each patient. lectin, alternative. three pathways merge at the level of complement component C3 activation. The complement component C4 plays a central role in the activation of the classic and lectin pathways. CFB is an activator of C3 and plays roles in alternative pathway. Our study shows elevated CFB is associated with worse neurological decits both at stroke onset and 3 months after acute ischemia. It suggests that alternative pathway may play an important role in secondary damage of stroke. Complement system is hyperactive in stroke process, especially in severe cases. CFB may be a good predictor of stroke severity. According to our study, elevated C3 is associated with severe symptom in early stage of stroke. Elevated C4 is associated with worse short-term prognosis. Combined previous studies, activation of complement system is correlated with severity and worse prognosis of stroke.


Background
Ischemic stroke is one of the leading causes of disability and mortality worldwide, and its incidence is increasing [1]. Recent years, in ammation has been shown to play an important role in the pathophysiology of ischemic stroke [2,3]. Progression of symptomatic intracranial large vessel disease is also associated with a proin ammatory state [4]. Following an acute focal brain injury during stroke, neural cell death orchestrates a secondary immune response characterized by glial activation, recruitment of peripheral immune cells, and release of cytokines and chemokines [3]. While post-stroke in ammation may contribute to the clearance of tissue debris and tissue repair, most published literature indicates that in ammation in the brain during the acute phase of stroke promotes the expansion of stroke lesions and worsens neurode cits [5,6].
Several studies using animal models suggests that immune cells such as lymphocytes, NKT cells, NK cells may be recruited several hours after stroke onset [7,8]. Amount of reports suggest that T lymphocytes play very crucial roles in the tissue damage following cerebral ischemia [9,10]. However, whether T cells exacerbate or alleviate the symptoms of stroke remains controversial [11,12].Higher NK cells counts was reported to be associated with post-stroke infection [13]. Some studies report that immunosuppression after stroke may cause infection and aggravate patients situation. Complement system were also found to play important roles in acute ischemic stroke [14]. However, what roles complement system and immunocytes played in the progression of ischemic stroke is still unclear.
To date, there are limited data about the effects of lymphocytes and complements on cerebral stroke severity and prognosis. Accordingly, the purpose of the present study was to access the association of plasma complements, T cell, NK cell and cerebral stroke severity in clinical cases.

Results
Demographics and characteristics of participants A total of 236 patients with acute ischemic stroke were included in our study. Baseline characteristics were shown in Table 1. The median age of all patients was 69 (58, 75) years old. 158 (66.95%) patients were male and the ratio of male to female was about 7:3. 173 (73.31%) patients had hypertension. 55 (23.31%) patients had diabetes. NIHSS score was 1.0 (1.0, 6.0). MRS score was 1.0 (0, 2.0). All the patients were categorized as mild (NIHSS score 0-4), moderate (NIHSS score 5-10), and severe group (NIHSS score ≥ 11)according to NHISS score at admission [15]. The characteristics of the patients are presented in Table 2. Gender (p = 0.176), prevalence of hypertension (p = 0.510) and diabetes (p = 0. 831) showed no signi cant difference in three groups. Age showed signi cant difference between severe group and other two groups. The patients in severe group are older than mild group (p = 0. 004) and moderate group (p = 0. 026). Age is not signi cantly different between mild group and moderate group (p = 0. 539).  Table 2 shows comparison of each immune factors in three groups. Statistical analysis indicated that NK cell(p = 0. 015), CFB (p = 0. 007), C3 (p = 0. 035) were signi cantly elevated in groups with higher NHISS score. CD4 + T cell (p = 0. 009), T cell (p = 0. 001) are decreased in groups with higher NHISS score. There was no signi cant difference on C4, CD8 + T cell and B cell. Results of post-hoc analysis was list in Table 2.
Association of immunocytes, complements and stroke severity In multivariate logistic regression (Table 3), CFB, C3 were independently associated with severity of stroke adjusting for age, gender, hypertension, diabetes. CD4 + T cell, NK cell, T cell were independently associated with severe ischemic stroke. All the patients were categorized as mild (mRS 0-2) and severe group (mRS > 2) according to mRS score at 3 months after stroke onset. The characteristics of the patients are presented in Table 4. Gender (p = 0.086), prevalence of hypertension (p = 0.573) and diabetes (p = 0. 993) showed no signi cant difference in three groups. The patients in severe group are signi cantly older than mild group (p = 0. 003). Comparison of immunocytes and complements Table 4 shows comparison of each immune factors in two groups according to mRS score. NK cell (p = 0.049), CFB (p = 0.035), C4 (p = 0.028) are were signi cantly elevated in groups with higher mRS score.

Discussion
It is known that the central nervous system actively interacts with the immune system, both directly and via intermediates [16]. First damage of ischemic stroke is caused by occlusion of artery and necrosis of neurons. Meanwhile, immune cells play critical roles in secondary damage after stroke onset [17]. A lot of studies elucidate that NK cells, lymphocytes, C3 and C4 and other immune factors participate in the development and destabilization of atherosclerotic plaques [18][19][20].T cells and NK cells were also found to recruit and in ltrate in the ischemic hemisphere [21,22,20]. Some studies proposed that ischemic stroke can induce immune suppression which may cause infection [23]. However, some other research found that immune cells aggravate the situation of infarct [12]. Inhibiting immune system activity could attenuate ischemic brain injury.
Our study found that elevated circulating CFB, C3 were independently associated with severity of stroke at onset. CD4 + T cell, NK cell, T cell were independently associated with severe ischemic stroke. CFB, C4 were independently associated with prognosis at 3 months.
The complement system is an essential part of innate immunity, typically conferring protection via eliminating pathogens and accumulating debris. Previous studies found that the level of C3 in plasma was higher in ischemic stroke patients than that in the healthy controls [24][25][26]. Elevated C3 in plasma from embolic ischemic stroke or cryptogenic stroke patients was associated with worse neurological outcomes 3 months and 2 years after ischemia onset [27,28,7]. Complement inhibition could improve the outcomes of ischemic stroke in many animal models [29]. There are limited clinical studies about stroke and C4 or CFB.
The complement system consists of three different activation pathways: classic, lectin, and alternative. All three pathways merge at the level of complement component C3 activation. The complement component C4 plays a central role in the activation of the classic and lectin pathways. CFB is an activator of C3 and plays roles in alternative pathway. Our study shows elevated CFB is associated with worse neurological de cits both at stroke onset and 3 months after acute ischemia. It suggests that alternative pathway may play an important role in secondary damage of stroke. Complement system is hyperactive in stroke process, especially in severe cases. CFB may be a good predictor of stroke severity. According to our study, elevated C3 is associated with severe symptom in early stage of stroke. Elevated C4 is associated with worse short-term prognosis. Combined previous studies, activation of complement system is correlated with severity and worse prognosis of stroke.
NK cells are part of the innate immune system. They control CNS in ammation by killing proin ammatory microglial cells, which are activated within minutes of ischemia onset [30]. Sylvie De Raedt [31] found the number of circulating NK cells within the rst hour after stroke was elevated, especially in patients with pneumonia later. Lünemann [30] found that NK cells catalyzed neuronal death and determined the infarct size. Our study suggest that elevated circulating NK cells are associated with severe stroke. Combined above studies, we suggest that elevated circulating NK cells count may be a potential indicator of severe stroke.
T lymphocytes are part of the adaptive immune system and are divided to several subtypes depending on their functional properties [12]. Previous studies found that stroke induced a dramatic and immediate loss of T-lymphocytes, which contributed to the stroke-induced immunosuppression. Immunosuppression can induce susceptibility to infection, which may aggravate the symptom and prognosis of stroke. There are also study reporting that CD4 + T cell loss may emerge as a predictive marker for post-stroke infection allowing and early identi cation of patients at risk [11]. Combined above studies, we suggest that loss of T cell and CD4 + T cell may be potential indicators of severe stroke.
As we know, complements, T cells, NK cells, B cells are all parts of immune system. In our study, we found NK cells, complements were positively correlated with severity and prognosis of stroke. However, T cells were negatively correlated with severity and prognosis of stroke. The results reveal the complexity of the role of the immune system in the pathogenesis of ischemic stroke. Immunocyte and immune factors play different roles in the course of stroke, which need more studies to discover.
In our study, we also found that age was correlated with severity and prognosis of stroke in the analysis of baseline characteristics. Older patients have a poor prognosis, which is consistent with our common clinical knowledge.
Some study limitations should be considered. Since our series is from a single medical center, small study population might have introduced statistical error. We test immune cells and complements, which cannot elucidate the immune mechanisms of stroke comprehensively. Further in ammatory cytokines and animal models are expected. More studies are needed to elucidate the molecular and cellular mechanisms of how complement components exert their functions in different stages of ischemic stroke to optimize the intervention of targeting the complement system.

Conclusion
Stroke triggers a robust and sustained shift in systemic immunity in patients. Our study found that elevated circulating CFB was correlated with both severe symptom at stroke onset and worse prognosis. It indicates that CFB may be a predictor of stroke severity and prognosis. Reduced T cell counts, elevated C3 and NK cell were associated with severe symptom at stroke onset. C4 was associated with short-term prognosis.

Consent for publication
Written informed consent for publication was obtained from all participants.

Competing interest
The authors declare that they have no competing interests.
Availability of data and material The datasets used or analysed during the current study are available from the corresponding author on reasonable request.