Immune cells can significantly predict and affect the clinical outcome of stroke. In particular, the neutrophil-to-lymphocyte ratio was shown to predict hemorrhagic transformation and the clinical outcome of stroke; however, the immunological mechanisms underlying these effects are poorly understood. Neutrophils are the first cells to invade injured tissue following focal brain ischemia. In these conditions, their proinflammatory properties enhance tissue damage and may promote ischemic incidences by inducing thrombus formation. Therefore, they constitute a potential target for therapeutic approaches and prevention of stroke. Indeed, in animal models of focal brain ischemia, neutrophils have been targeted with successful results. However, even in brain lesions, neutrophils also exert beneficial effects, because they are involved in triggering immunological removal of cell debris. Furthermore, intact neutrophil function is essential for maintaining immunological defense against bacterial infections. Several studies have demonstrated that stroke-derived neutrophils displayed impaired bacterial defense capacity. Because infections are known to impair the clinical course of stroke, therapeutic interventions that target neutrophils should preserve or even restore their function outside the central nervous system (CNS). This complex situation requires well-tailored therapeutic approaches that can effectively tackle immune cell invasion in the brain but avoid increasing poststroke infections.

Background and Purpose—: Stroke-induced immune alterations predispose patients to infections. Although the relationship between stroke and the adaptive immune system has been investigated in detail, to date it is unknown whether the innate immune system, which forms the first line of antibacterial defense, is also impaired in patients with stroke. Therefore, we investigated whether chemotaxis, phagocytosis, oxidative burst, degranulation of defensins, and NETosis in monocytes and in neutrophil granulocytes are altered in patients with stroke compared with controls. Methods—: Sixty-three patients having acute ischemic stroke were recruited within 12 hours of symptom onset; blood was sampled on admission and on days 1, 3, 5, and 7. Thirty-seven age-matched controls were also recruited. Cell migration, phagocytosis, and oxidative burst of phagocytes were determined in vitro. Human neutrophil peptides 1 to 3 and serum metanephrine levels were measured by enzyme-linked immunosorbent assay, and NETosis was quantified by immunohistochemistry. Results—: The key mechanisms required for bacterial killing, oxidative burst, and NETosis were significantly reduced in samples taken from patients with stroke compared with controls, whereas migration, phagocytic function, and defensin production remained unimpaired in monocytes and granulocytes from patients with stroke. Conclusions—: Stroke-induced immune alterations include impairment of the first-line defense performed by specialized phagocytes against bacteria. The hypothesis that these changes enhance susceptibility to acquired infections is supported by our observation that on admission oxidative burst in monocytes was more impaired in patients with stroke with subsequent stroke-associated infections.

Background and Purpose. Regulatory T cells (Tregs) have been suggested to modulate stroke-induced immune responses. However, analyses of Tregs in patients and in experimental stroke have yielded contradictory findings. We performed the current study to assess the regulation and function of Tregs in peripheral blood of stroke patients. Age dependent expression of CD39 on Tregs was quantified in mice and men. Methods. Total FoxP3⁺ Tregs and CD39⁺FoxP3⁺ Tregs were quantified by flow cytometry in controls and stroke patients on admission and on days 1, 3, 5, and 7 thereafter. Treg function was assessed by quantifying the inhibition of activation-induced expression of CD69 and CD154 on T effector cells (Teffs). Results. Total Tregs accounted for 5.0% of CD4⁺ T cells in controls and <2.8% in stroke patients on admission. They remained below control values until day 7. CD39⁺ Tregs were most strongly reduced in stroke patients. On day 3 the Treg-mediated inhibition of CD154 upregulation on CD4⁺ Teff was impaired in stroke patients. CD39 expression on Treg increased with age in peripheral blood of mice and men. Conclusion. We demonstrate a loss of active FoxP3⁺CD39⁺ Tregs from stroke patient’s peripheral blood. The suppressive Treg function of remaining Tregs is impaired after stroke.

Advances in spine surgery enable technically safe interventions in older patients with disabling spine disease, yet postoperative delirium (POD) poses a serious risk for postoperative recovery. This study investigates biomarkers of pro-neuroinflammatory states that may help objectively define the pre-operative risk for POD. This study enrolled patients aged ≥60 scheduled for elective spine surgery under general anesthesia. Biomarkers for a pro-neuroinflammatory state included S100 calcium-binding protein β (S100β), brain-derived neurotrophic factor (BDNF), Gasdermin D, and the soluble ectodomain of the triggering receptor expressed on myeloid cells 2 (sTREM2). Postoperative changes of Interleukin-6 (IL-6), Interleukin-1β (IL-1β), and C-reactive protein (CRP) were assessed as markers of systemic inflammation preoperatively, intraoperatively, and early postoperatively (up to 48 h). Patients with POD (n = 19, 75.7 ± 5.8 years) had higher pre-operative levels of sTREM2 (128.2 ± 69.4 pg/mL vs. 97.2 ± 52.0 pg/mL, p = 0.049) and Gasdermin D (2.9 ± 1.6 pg/mL vs. 2.1 ± 1.4 pg/mL, p = 0.29) than those without POD (n = 25, 75.6 ± 5.1 years). STREM2 was additionally a predictor for POD (OR = 1.01/(pg/mL) [1.00–1.03], p = 0.05), moderated by IL-6 (Wald-χ² = 4.06, p = 0.04). Patients with POD additionally showed a significant increase in IL-6, IL-1β, and S100β levels on the first postoperative day. This study identified higher levels of sTREM2 and Gasdermin D as potential markers of a pro-neuroinflammatory state that predisposes to the development of POD. Future studies should confirm these results in a larger cohort and determine their potential as an objective biomarker to inform delirium prevention strategies.

Background: Newborns are prone to infections, which are independent predictors of neonatal mortality and morbidity. Neutrophil extracellular traps (NETs) are structures composed of chromatin and antimicrobial molecules that capture and kill pathogens. NETs may play an important role in the innate immune system and, thus, might be associated with impaired neonatal immune function. Objectives: This study aimed to compare NET formation between term neonates and healthy adults. We additionally investigated the effects of gestational age, birth weight, mode of delivery, gender, and perinatal infections. Methods: We collected cord blood from 57 term infants (mean gestational age, 39.1 weeks) and 9 late preterm infants (35 weeks), and peripheral blood from 18 healthy adult donors. Neutrophils were isolated, and then NET formation was induced using three different stimulants: N-formylmethionine-leucyl-phenylalanine, phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide. NETs were immunohistochemically stained and analyzed with regard to NET percentage and NET area. Results: With all three stimuli, healthy term infants showed a lower NET percentage than the adult control group (p < 0.0001 each). The groups also differed in NET area, but the significance level was lower. Following PMA stimulation, we observed greater reductions in NET percentage and NET area in preterm than term infants. Conclusions: The lower NET formation observed in term infants compared to adults likely contributes to the reduced neonatal immune response. NET formation appeared to be even further decreased in late preterm neonates. There remains a need for further investigations of NET formation in more immature preterm infants.

Stroke induces immune alterations such as impaired oxidative burst and reduced release of neutrophil extracellular traps (NETs). We hypothesised that key enzymes of these defence mechanisms may be altered in ischaemic stroke. Therefore, we analysed the intra- and extracellular amounts of myeloperoxidase (MPO) and neutrophil elastase (NE) in patient sera and granulocytes and monocytes. Because the autonomous nervous system is thought to mediate stroke-induced immune alterations, we also studied the influence of stress hormones and acetylcholine on MPO and NE. Rapid recanalization by recombinant tissue plasminogen activator (r-tPA) is the only available treatment for ischaemic stroke besides thrombectomy, and its influence on antibacterial defence mechanisms of granulocytes and monocytes were addressed here. Ex vivo: Intracellular and serum MPO and NE were measured on days 0, 1, 3 and 5 post-stroke by either flow cytometry or enzyme-linked immunosorbent assay (ELISA) and compared to controls. In vitro: Blood from healthy donors was incubated with catecholamines, dexamethasone and acetylcholine, and the percentage of NET-producing cells and the area covered by NETs were quantified immunohistochemically. Intra- and extracellular MPO and NE were quantified by flow cytometry or ELISA. Blood samples from healthy donors were incubated with r-tPA, and oxidative burst, phagocytosis, NETosis, cytokine release, MPO and NE were quantified by flow cytometry, ELISA and microscopy. MPO was reduced in granulocytes but increased in sera obtained from stroke patients compared to controls. NE was not altered intracellularly but was elevated in patient sera. The percentage of NET-producing neutrophils was decreased by stress hormones and increased by acetylcholine. Neither intracellular MPO nor NE was altered by hormone treatment; however, adrenaline and acetylcholine induced NE release. r-tPA led to reduced phagocytosis and oxidative burst in granulocytes and monocytes in vitro. NETosis, MPO release and cytokines were not altered, whereas NE release was enhanced by r-tPA. Intracellular reduction of MPO might be responsible for reduced NETosis in stroke patients. The impact of enhanced MPO and NE serum levels in stroke patients should be addressed in future studies. r-tPA impaired antibacterial defence function in vitro. Therefore, patients who undergo unsuccessful recanalization therapy might be at higher risk for infection, which should be analysed in future investigations. Immune alterations due to r-tPA effects in stroke patients should also be investigated.