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Ischemic stroke profoundly influences the peripheral immune system, which responds quickly to brain ischemia and participates in the evolution of poststroke neuroinflammation, while a period of systemic immunosuppression ensues. Poststroke immunosuppression brings harmful consequences, including increased infection rates and escalated death. As the most abundant cell population in the fast-responding innate immune system, myeloid cells including neutrophils and monocytes play an indispensable role in systemic immunosuppression after stroke. The change in myeloid response after stroke can be regulated by circulating DAMPs (damage-associated molecular patterns) and neuromodulatory mechanisms, which contain sympathetic nervous system, hypothalamic-pituitary-adrenal, and parasympathetic nervous system. In this review, we summarize the emerging roles and newly identified mechanisms underlying myeloid cell response in poststroke immunosuppression. Deeper understanding of the above points may pave the way for future development of novel therapeutic strategies to treat poststroke immunosuppression.

Immune cell infiltration to the injured brain is a key component of the neuroinflammatory response after ischemic stroke. In contrast to the large amount of proinflammatory immune cells, regulatory T cells, are an important subgroup of T cells that are involved in maintaining immune homeostasis and suppress an overshooting immune reaction after stroke. Numerous previous reports have consistently demonstrated the beneficial role of this immunosuppressive immune cell population during the acute phase after experimental stroke by limiting inflammatory lesion progression. Two recent studies expanded now this concept and demonstrate that regulatory T cells-mediated effects also promote chronic recovery after stroke by promoting a proregenerative tissue environment. These recent findings suggest that boosting regulatory T cells could be beneficial beyond modulating the immediate neuroinflammatory response and improve chronic functional recovery.

Introduction: The gut microbiota influences the severity of brain injury through regulation of intestinal immune homeostasis. However, molecular cues involved in microbiota-immune interaction after stroke are unknown. Metabolites have a pivotal role in connecting commensals to the immune response. Specifically, bacteria-derived metabolites of tryptophan catabolism activate the transcription factor aryl hydrocarbon receptor (AhR) which regulates T cell polarization directly or via tolerogenic function of dendritic cells (DCs). Hypothesis: Since adaptive immune cells are involved in stroke, we hypothesize that modulation of tryptophan metabolism–orchestrated by bacteria–modifies T cell differentiation and influences stroke outcome. Methods: Stroke is induced by the occlusion of the middle cerebral artery (MCAO) in C57BL6 mice. Blood and the distal small intestine are sampled for the detection of tryptophan metabolites and quantified by metagenomics or mRNA expression. Immune cells are isolated from the intestine and analyzed by flow cytometry. Stroke outcome is measured by infarct volumetry and behaviour tests. Results: Tryptophan catabolism was augmented in mice subjected to stroke-induced dysbiosis–shown by a) an increase of the most proximal metabolite kynurenine and a concomitant decrease of tryptophan and b) an upregulation of mRNA expression of the rate-limiting enzyme involved in tryptophan’s breakdown. This was paralleled by an increase of AhR mRNA in the ileum after MCAO. To test the implication of AhR on the tolerogenic function of DCs, we used AhR-DCs knock-out mice. We found that CD103+CD11b+ DCs lacking AhR were downregulated after stroke compared to wild-type (WT) mice. Interestingly, polarization state of T cells was modified in DCs lacking AhR (regulatory versus Th17 cell ratio). This could influence the central nervous system immune response and induce protection after stroke, as suggested by a reduction of infarct volume observed 3 days after stroke in AhR-DCs knock-out mice compared to WT mice. Conclusion: These findings suggest that stroke-induced dysbiosis leads to an increase of tryptophan catabolism, and that AhR depletion in DCs influences immune cell polarization and stroke outcome.

Background and Purpose— Neuroinflammation plays an important role in ischemic brain injury. Regulatory T cells (Treg) are important endogenous immune modulators. We tested the hypothesis that Treg amplification with a CD28 superagonistic monoclonal antibody (CD28SA) reduces brain damage in murine cerebral ischemia. Methods— Cerebral ischemia was induced by coagulation of the distal middle cerebral artery or by 60 minutes filament occlusion of the proximal middle cerebral artery in C57BL6 mice. 150 μg CD28SA was injected intraperitoneally 3 or 6 hours after ischemia onset. Outcome was determined by infarct volumetry and behavioral testing. Brain-infiltrating leukocyte subpopulations were analyzed by flow cytometry and immunohistochemistry 3 and 7 days after middle cerebral artery occlusion. Results— CD28SA reduced infarct size in both models and attenuated functional deficit 7 days after stroke induction. Mice treated with CD28SA increased numbers of Treg in spleen and brain. Tregs were functionally active and migrated into the brain where they accumulated and proliferated in the peri-infarct area. More than 60% of brain infiltrating Treg produced interleukin-10 in CD28SA compared with 30% in control. Conclusions— In vivo expansion and amplification of Treg by CD28SA attenuates the inflammatory response and improves outcome after experimental stroke.

Background and Purpose— Neuroinflammatory processes contribute to secondary neuronal damage after intracerebral hemorrhage. We aimed to characterize the time course of brain immigration of different leukocyte subsets after striatal injection of either autologous blood or collagenase in mice. Methods— Intracerebral hemorrhage was induced by injection of either autologous blood (20 μL) or collagenase (0.03 U) in C57Bl/6J mice. Hematoma volumetry was performed on cryosections. Blood volume was measured by hemoglobin spectrophotometry. Leukocytes were isolated from hemorrhagic hemisphere 1, 3, 5, and 14 days after intracerebral hemorrhage, stained for leukocyte markers, and measured by flow cytometry. Heterologous blood injection from CD45.1 mice was used to investigate the origin of brain-invading leukocytes. Results— Collagenase injection induced a larger hematoma volume but a similar blood content compared with blood injection. Cerebral leukocyte infiltration in the hemorrhagic hemisphere was similar in both models. The majority of leukocytes isolated from the brain originated from the circulation. CD4 + T lymphocytes were the predominant brain leukocyte population in both models. However, cerebral granulocyte counts were higher after collagenase compared with blood injection. Conclusions— Brain infiltration of systemic immune cells is similar in both murine intracerebral hemorrhage models. The pathophysiological impact of invading leukocytes and, in particular, of T cells requires further investigation.

Background and Purpose— Cerebral ischemia is ensued by a cellular immune depression syndrome. The postischemic functional capacity of T lymphocytes is controversial, and interactions between leukocyte subsets are largely unknown. Understanding the immunologic interplay between antigen-presenting cells and lymphocytes as well as between distinct lymphocyte subsets after stroke might be of clinical/therapeutic significance because animal data argue for a cerebroprotective effect of, for example, CD4+CD25+ regulatory T cells. Methods— Ex vivo CD4+ T cell proliferation was analyzed in experimental and human stroke using fluorescence activated cell sorter analysis. To investigate suppressive effects of CD4+CD25+ regulatory T cells as well as the influence of costimulatory cells on CD4+ T cell proliferation, subsets were magnetically sorted before proliferation assay setup. Results— After stroke: (1) proliferation of mouse and human CD4+ T cells on T cell receptor stimulation was unaltered; (2) the suppressive effect of CD4+CD25+ regulatory T cells in mouse and man was unaltered; and (3) efficacy of circulating costimulatory cells from stroke animals was reduced by a mean of 0.6 (SEM 0.1, P =0.001) CD4+ T cell division numbers compared with sham-treated animals. Conclusions— Reduced costimulatory efficacy of circulating costimulatory cells in mice is an important feature of stroke-induced immunodepression. Understanding the interplay of costimulatory cells and responder T cells (eg, CD4+ T cells or CD4+CD25+ regulatory T cells) after stroke may offer new insights into the prevention of secondary inflammatory damage to the brain and help to guide new therapeutic strategies.

In their insightful and stimulating comment on our article1 and on the persistent controversy over the pathophysiological role of regulatory T cells in general, Gauberti and Vivien address perspectives and potential pitfalls of experimental stroke research in the field of neuroinflammation. Obviously, we share their overall view that there is potential for immunomodulatory therapies, including amplification of regulatory T cells, that limit early inflammatory cytoxicity after stroke. Specifically, Gauberti and Vivien make the convincing argument that the most widely used stroke model, transient mechanical vascular occlusion of the middle cerebal artery (TMVO), may induce pathophysiological processes, including thromboinflammation, that are unlikely to play a role in other models and in the majority of strokes in patients. General scepticism toward the translational validity of the …

Response: We appreciate the interest of Dr Moll1 in our experimental study in which we examined the effect of different hemostatic approaches on early hematoma growth in mice treated with dabigatran etexilate. In contrast to another recently published article2 using a similar experimental model, treatment with high doses of dabigatran in our study induced an excess early hematoma growth compared with nonanticoagulated mice, which we documented by serial MRI. Moreover, we showed that prothrombin complex (PCC) was the most consistently effective agent preventing the excess hematoma growth in mice treated with either 4.5 mg/kg or 9 mg/kg of dabigatran etexilate intraperitoneally. Finally, we tested the effect of 3 different doses of PCC on …

Background and Purpose— Dabigatran-etexilate (DE) recently has been approved for stroke prevention in atrial fibrillation. However, lack of effective antagonists represents a major concern in the event of intracerebral hemorrhage (ICH). The aims of the present study were to establish a murine model of ICH associated with dabigatran, and to test the efficacy of different hemostatic factors in preventing hematoma growth. Methods— In C57BL/6 mice receiving DE (4.5 or 9.0 mg/kg), in vivo and in vitro coagulation assays and dabigatran plasma levels were measured repeatedly. Thirty minutes after inducing ICH by striatal collagenase injection, mice received an intravenous injection of saline, prothrombin complex concentrate (PCC; 100 U/kg), murine fresh-frozen plasma (200 μL), or recombinant human factor VIIa (8.0 mg/kg). ICH volume was quantified on brain cryosections 24 hours later. Results— DE substantially prolonged tail vein bleeding time and ecarin clotting time for 4 hours corresponding to dabigatran plasma levels. Intracerebral hematoma expansion was observed mainly during the first 3 hours on serial T2* MRI. Anticoagulation with high doses of DE increased the hematoma volume significantly. PCC and, less consistently, fresh-frozen plasma prevented excess hematoma expansion caused by DE, whereas recombinant human factor VIIa was ineffective. Prevention of hematoma growth and reversal of tail vein bleeding time by PCC were dose-dependent. Conclusions— The study provides strong evidence that PCC and, less consistently, fresh-frozen plasma prevent excess intracerebral hematoma expansion in a murine ICH model associated with dabigatran. The efficacy and safety of this strategy must be further evaluated in clinical studies.

Background and Purpose— Therapeutic modification of the postischemic immune processes is a key target of current experimental stroke research. For successful translation into the clinical setting, experimental studies must account for the impact of different strokes on the immune system including susceptibility to infection. Herein, we characterize the impact of 3 ischemia models on systemic immunological and microbiological parameters. Methods— In C57Bl/6 mice (n=235), the middle cerebral artery was occluded (MCAO) either permanently by distal coagulation or transiently by an intraluminal filament for 30 minutes or 90 minutes. Differential leukocyte counts were performed in blood and lymphatic organs. Lymphocyte subpopulations and apoptotic cells were characterized by flow cytometry. Blood cytokine concentrations were measured by ELISA. Microbiological cultures were grown from blood and lung samples. Results— Only extensive infarcts induced leukopenia 24 hours, 3 days and 7 days after MCAO and decreased lymphocyte counts in spleen, lymph nodes and thymus. In contrast, small infarcts led to no significant changes in differential blood count or reduction of overall cell counts in lymphatic organs. Splenic lymphocyte apoptosis and blood cytokine production was significantly increased after extensive lesions compared to mild ischemia. Hypothermia and weight loss occurred only in mice with large infarcts which also suffered from pneumonia and sepsis. In contrast to infarct size, location and side of the infarct did not affect physiological parameters and immune cell alterations. Conclusions— Postischemic systemic immunomodulation and infectious complications differ substantially among stroke models. Translational studies of immunomodulatory therapies for stroke must account for this heterogeneity.