Ischemic stroke is a general term for necrosis of brain tissue caused by stenosis, occlusion, or insufficiency of arteries that supply blood to the brain (e.g., carotid and vertebral arteries). It accounts for approximately 87% of all strokes. Acute ischemic stroke (AIS) is a life-threatening disease with high morbidity and mortality worldwide (1–3). The pathophysiology of AIS involves immune cell activation, inflammation, and programmed cell death; of these, immune responses play a pivotal role given their contribution to both tissue damage and repair (1, 4, 5). More specifically, during the acute stage, ischemia-induced immune response damages the brain; in the sub-acute stage, Treg cells and macrophages protect and repair the brain (6–9).

Aging is an important factor that affects immune response, thereby influencing clinical outcomes (10, 11). Although the elderly are among the most vulnerable to AIS, the influence of aging and consequent changes to immune response in AIS has not been studied well. Theoretically, as people grow older, ischemia-induced immune response may be modified and weakened, which may result in poorer outcomes. To elucidate the impact of aging on immune response to AIS, we analyzed the alterations in immune cell composition in peripheral blood of AIS patients based on their age groups. The results of this study may become references for predicting outcomes and designing customized treatment.

The Ethics Committee of Xuanwu Hospital, Capital Medical University approved this study, and all patients signed informed consents prior to their enrollment.

Final analysis included data from 41 eligible patients, divided into group-A (n = 13), group-B (n = 18), and group-C (n = 10). Among the three groups, there were no statistically significant differences in stroke onset to door times (P > 0.05, Table 1).

Immune response after AIS is a well-described phenomenon that affects clinical outcomes. It is now widely accepted that inflammation and immune mediators are potential therapeutic targets for AIS treatment due to their critical role in inducing acute neuronal damage post-AIS (1, 14). However, targeting inflammation and immune mediators are not straightforward as immune responses change with increasing age. In this study, we revealed for the first time that aging alters immune cell activity and composition in the peripheral blood of patient post-AIS. Our results offer an important reference for predicting outcomes and designing customized treatments.

During the aging process, the human immune system remodels and degenerates, which heavily impacts health outcomes (15). Both innate and adaptive immune systems' functions decline with age, which contribute to the development of age-related diseases such as neurodegenerative and cerebral vascular diseases (16, 17). The major immune cell types including monocytes, natural killer (NK) cells, dendritic cells, B lymphocytes, and T lymphocytes have been reported to play important roles in the pathogenesis of stroke (18). Since aging affects almost all kinds of immune cells, (19) we studied the alterations in immune cell composition in peripheral blood of acute ischemic stroke (AIS) patients based on their age group. The results may become a reference for future research, construction of predictive models, as well as designing of customized treatment. Previous studies have regarded AIS patients younger than 55 as young or middle-aged adults, while patients over 65 were regarded as elderly; these two populations possess distinct ischemic stroke risk factors and outcomes (12, 13, 20). In accordance to the distinct sets of risks and outcomes, our study revealed that aging affects the composition of peripheral blood immune cells.

NK cells are a vital subset of immune cells in ischemic brains. NK cells have been reported to be associated with inflammation, immunodepression, and infections post-stroke, indicating that they play multiple complex roles in stroke pathogenesis (21). Two conventional NK cell subsets, CD56⁻CD16⁺ NK-cells and CD56⁺CD16⁻ NK-cells, which are widely distributed in human peripheral blood and lymphoid tissues, were assayed in the present study. The results showed that the proportion of CD56⁺CD16⁻ NK-cells was the lowest in the age group including 55–65-year-olds, while the proportion of CD56⁻CD16⁺ NK-cells increased with aging. These results suggest that NK-cells-mediated immunosuppression and inflammation deteriorates with increasing age, for which future therapeutic strategies for the elderly should make appropriate compensations.

T-cells consist of several subsets expressing various combinations of CD3, CD4, and/or CD8 molecules, thereby functioning in diverse capacities in order to regulate immunological and inflammatory homeostasis. T-cells have been identified in brain tissue as early as 24 h after ischemia. In ischemic brain tissue, CD3⁺ T-cells were found to perform a critical role in permitting susceptibility to brain tissue injury in the early phase of ischemic stroke (22, 23). In the meantime, CD4⁺ T lymphocytes mediated the production of interleukins, tumor necrosis factor and other inflammatory factors, while CD8⁺ T lymphocytes participated in neuronal injury by releasing cytotoxic proteases (24). Both CD4⁺ and CD8⁺ T-cells aggravated brain ischemia/reperfusion injury via inflammatory and thrombogenic responses, resulting in larger infarct volumes and greater neurological deficits (25). Furthermore, CD3⁺CD4⁻CD8⁻ T-cell (double-negative T-cells, DNT) counts increased in both peripheral blood and ischemic brain tissue in patients with AIS. The infiltrating DNTs played a critical role in promoting microglia-mediated neuroinflammation and ischemic brain injury (26). In the present study, CD3+ T-cells diminished with age: The proportion of CD3⁺ T-cells in group-C (age above 65-year-old) was significantly lower than that of group-A. The CD3⁺CD4⁺ T-cells also significantly decreased with age in group-C when compared to group-A and group-B. Meanwhile, CD3⁺CD4⁻CD8⁻ T-cells and CD3⁺CD8⁺ T-cells increased with age. These results suggest that T-cell mediated immune responses in AIS were more active in younger patients when compared to the elderly population.

Monocytes present heterogeneously with proinflammatory or anti-inflammatory phenotypes depending on their stage of differentiation and the mechanism by which they were activated. Monocytes reach the central nervous system as early as 4 h from the onset of AIS. Monocytes consist of several subsets such as CD14highCD16⁻ monocytes, CD14highCD16⁺ monocytes, and CD14dimCD16⁺ monocytes; each colony of monocytes have different time courses in AIS patients and are associated with distinct clinical patterns (27). Clarifying the role of each monocyte subsets in the pathogenesis of AIS would be helpful for designing early interventions in the future. The present study found that the proportion of total monocytes nor any subset of monocytes varied significantly among the three groups. These results reveal that aging may not have significant effects on monocyte response to AIS.

B-cells are a part of the adaptive immune system and are responsible for many immunological functions. Previous studies reported that B cells did not contribute to the acute stage of stroke pathophysiology (28). Rather, B-cells were found participating in stroke recovery 4-days after stroke onset by directly protecting neurons and promoting recovery within the injured and reorganizing brain (29). The present study revealed that CD19⁺ B-cell concentrations did not vary among different age groups.

In addition, peripheral blood immune cells highly involved in brain tissue damage through stimulating inflammatory factors production in the early onset of ischemia pathological process. In order to investigate the inflammatory biomarkers' effect on the immune response whether depended on different age groups, we further analyzed the relevance between the white blood cells count, neutrophils count, hypersensitive c-reactive protein concentration with immune cells. Results showed that white blood cells count, neutrophils count and hypersensitive c-reactive protein concentration has no obvious difference between the 3 groups, thus proved the inflammatory biomarkers between the three groups has no obvious correlation with the compositions of immune cells of AIS patients. Although the percentage of CD3⁺ T cells and CD3⁺ CD4⁺ T cells may have potential correlation with the white blood cells count, but this result did not reach statistical significance obviously, still calling for further study.

Limitations of this study were as follows: Firstly, this was a single-center, real-world cohort study, making it susceptible to systematic errors. Secondly, the number of cases was small and without multi-timepoint analysis. The benefits of extending this study to more people and multiple timepoints did not outweigh the possible ethical concerns.

The distributions of immune cells in patients with AIS in different ages were variable. The proportion of NK-cells was the lowest in patients aged 55–65 years. Ratios of both CD3⁺ T-cells and CD3⁺CD4⁺ T-cells decreased with greater age. Monocytes' and B-cells' distribution patterns did not vary significantly across the age groups, thus it may be possible that they do not greatly influence clinical outcomes of AIS. Any of the aforementioned results may impact clinical outcomes and should be considered in customized treatment.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving human participants were reviewed and approved by Ethics Committee of Xuanwu Hospital, Capital Medical University. The patients/participants provided their written informed consent to participate in this study.

RM, HZ, HL, and YD drafted and revised the manuscript. RM and HZ conceptualized, designed the study, and approved the final manuscript. HZ collected, assembled, interpreted the data, and drew the figures. JG, CW, KD, ZL, LC, and HS collected the data. All authors contributed to the article and approved the submitted version.

This work was supported by the National Key R&D Program of China under Grant 2017YFC1308400; the National Natural Science Foundation under Grants 81801143, 82171297, and 82101390; the Beijing Natural Science Foundation 7212047.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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