Juliane Klehmet

Stroke-Induced Immunodepression Experimental Evidence and Clinical Relevance

Stroke affects the normally well-balanced interplay of the 2 supersystems: the nervous and the immune system. Recent research elucidated some of the involved signals and mechanisms and, importantly, was able to demonstrate that brain-immune interactions are highly relevant for functional outcome after stroke. Immunodepression after stroke increases the susceptibility to infection, the most relevant complication in stroke patients. However, immunodepression after stroke may also have beneficial effects, for example, by suppressing autoaggressive responses during lesion-induced exposure of central nervous system-specific antigens to the immune system. Thus, before immunomodulatory therapy can be applied to stroke patients, we need to understand better the interaction of brain and immune system after focal cerebral ischemia. Until then, anticipating an important consequence of stroke-induced immunodepression, bacterial infection, preventive antibiotic strategies have been proposed. In mouse experiments, preventive antibiotic treatment dramatically improves mortality and outcome. Results of clinical studies on this issue are contradictory at present, and larger trials are needed to settle the question whether (and which) stroke patients should be preventively treated. Nevertheless, clinical evidence is emerging demonstrating that stroke-induced immunodepression in humans not only exists, but has very similar features to those characterized in rodent experiments.

Preventive Antibacterial Therapy in Acute Ischemic Stroke: A Randomized Controlled Trial

Background Pneumonia is a major risk factor of death after acute stroke. In a mouse model, preventive antibacterial therapy with moxifloxacin not only prevents the development of post-stroke infections, it also reduces mortality, and improves neurological outcome significantly. In this study we investigate whether this approach is effective in stroke patients. Methods Preventive ANtibacterial THERapy in acute Ischemic Stroke (PANTHERIS) is a randomized, double-blind, placebo-controlled trial in 80 patients with severe, non-lacunar, ischemic stroke (NIHSS>11) in the middle cerebral artery (MCA) territory. Patients received either intravenous moxifloxacin (400 mg daily) or placebo for 5 days starting within 36 hours after stroke onset. Primary endpoint was infection within 11 days. Secondary endpoints included neurological outcome, survival, development of stroke-induced immunodepression, and induction of bacterial resistance. Findings On intention-to treat analysis (79 patients), the infection rate at day 11 in the moxifloxacin treated group was 15.4% compared to 32.5% in the placebo treated group (p = 0.114). On per protocol analysis (n = 66), moxifloxacin significantly reduced infection rate from 41.9% to 17.1% (p = 0.032). Stroke associated infections were associated with a lower survival rate. In this study, neurological outcome and survival were not significantly influenced by treatment with moxifloxacin. Frequency of fluoroquinolone resistance in both treatment groups did not differ. On logistic regression analysis, treatment arm as well as the interaction between treatment arm and monocytic HLA-DR expression (a marker for immunodepression) at day 1 after stroke onset was independently and highly predictive for post-stroke infections. Interpretation PANTHERIS suggests that preventive administration of moxifloxacin is superior in reducing infections after severe non-lacunar ischemic stroke compared to placebo. In addition, the results emphasize the pivotal role of immunodepression in developing post-stroke infections. Trial Registration Controlled-Trials.com ISRCTN74386719

Stroke-induced immunodepression and post-stroke infections: Lessons from the preventive antibacterial therapy in stroke trial

UNLABELLED Infections are a leading cause of death in patients with acute CNS injury such as stroke. Recent experimental evidence indicated that stroke leads to suppression of innate and adaptive peripheral immune responses which predisposes to infection. However, less is known on phenotypic and functional immune alterations in correlation with the occurrence of infectious complications in patients with acute stroke. EXPERIMENTAL PROCEDURES In the recently completed randomized, double blind, placebo-controlled Preventive Antibacterial Therapy in Stroke (PANTHERIS) trial on the efficacy of short-term antibacterial therapy to prevent the development of post-stroke infections, we assessed longitudinal changes in lymphocyte subpopulations and mitogen-induced lymphocytic interferon gamma (IFN)-gamma production using flow cytometry in 80 patients with acute severe stroke at days 1, 3, 8, 90 and 180 after clinical onset. Plasma interleukin (IL)-6 and IL-10 concentration as well as urinary levels of norepinephrine and cortisol was assessed within the first 8 days after stroke. Patients of the placebo and verum (moxifloxacin) treatment groups who did or did not develop infections within 11 days after stroke were compared to identify immunological changes associated with the occurrence of post-stroke infections. RESULTS Rapid T-lymphopenia and long-lasting suppression of lymphocytic IFN-gamma production were observed in all stroke patients. Patients of the placebo group who developed infections showed a trend toward greater decline of CD4+ Th cell counts and higher urinary levels of norepinephrine early after stroke than patients without infections. Onset of infections was accompanied with higher plasma IL-6 levels in the placebo group but not in the moxifloxacin group. In addition, an early rise in plasma IL-10 was detected in patients who developed infections despite preventive antibacterial treatment. CONCLUSION A rapid loss and functional deactivation of T cells are common changes in stroke patients consistent with immunodepression after brain ischemia. A stronger decrease in cellular immune responses and an increased sympathetic activity after stroke are associated with a higher risk of infections. Increased plasma levels of the anti-inflammatory cytokine IL-10 early after stroke may identify patients who will not respond to preventive antibacterial therapy with moxifloxacin.

Regulatory T Cells Accumulate and Proliferate in the Ischemic Hemisphere for up to 30 Days after MCAO

Local and peripheral immune responses are activated after ischemic stroke. In our present study, we investigated the temporal distribution, location, induction, and function of regulatory T cells (Tregs) and the possible involvement of microglia, macrophages, and dendritic cells after middle cerebral artery occlusion (MCAO). C57BL/6J and Foxp3EGFP transgenic mice were subjected to 30 minutes MCAO. On days 7, 14, and 30 after MCAO, Tregs and antigen presenting cells were analyzed using fluorescence activated cell sorting multicolor staining and immunohistochemistry. A strong accumulation of Tregs was observed on days 14 and 30 in the ischemic hemisphere accompanied by the elevated presence and activation of microglia. Dendritic cells and macrophages were found on each analyzed day. About 60% of Foxp3+ Tregs in ischemic hemispheres were positive for the proliferation marker Ki-67 on days 7 and 14 after MCAO. The transfer of naive CD4+ cells depleted of Foxp3+ Tregs into RAG1−/– mice 1 day before MCAO did not lead to a de novo generation of Tregs 14 days after surgery. After depletion of CD25+ Tregs, no changes regarding neurologic outcome were detected. The sustained presence of Tregs in the brain after MCAO indicates a long-lasting immunological alteration and involvement of brain cells in immunoregulatory mechanisms.

Mouse Strains Differ in Their Susceptibility to Poststroke Infections

Objective: Severe infections, in particular pneumonia, have a major impact on the clinical management and outcome of stroke patients. In a mouse model we have recently demonstrated that stroke induces immunodepression which can result in life-threatening infections. Here, we investigated whether the susceptibility to infections after stroke is strain dependent. Methods and Results: Mice from 129SV, C57/B6, and Balb/C strains were subjected to experimental stroke by filament occlusion of the middle cerebral artery (MCAO) for 60 min. Infarct volumes were measured 3 days after MCAO. Microbiological assessment was based on cultures of bronchoalveolar lavage (BAL), lung tissue and blood of animals obtained 3 days after stroke. Three days after stroke 129SV mice did not only develop bacterial chest infection, but also had a strongly increased susceptibility to bacteremia. In contrast, C57BL/6 and Balb/C mice acquired bacterial lung infections only. In addition, bacterial load in BAL was significantly higher in 129SV mice than in the other mice strains. These differences in susceptibility to infection did not correlate with infarct volumes. Conclusions: Stroke-associated pneumonia developed in three commonly used mouse strains while severity of infections differed between strains. Since infections affect outcome, monitoring of infections is highly relevant for the interpretation of results in experimental stroke research.

Blocking Stroke-Induced Immunodeficiency Increases CNS Antigen-Specific Autoreactivity But Does Not Worsen Functional Outcome after Experimental Stroke

Stroke-induced immunodepression (SIDS) is an essential cause of poststroke infections. Pharmacological inhibition of SIDS appears promising in preventing life-threatening infections in stroke patients. However, SIDS might represent an adaptive mechanism preventing autoreactive immune responses after stroke. To address this, we used myelin oligodendrocyte glycoprotein (MOG) T-cell receptor transgenic (2D2) mice where >80% of peripheral CD4+ T cells express a functional receptor for MOG. We investigated in a murine model of middle cerebral artery occlusion the effect of blocking SIDS by inhibiting bodys main stress axes, the sympathetic nervous system (SNS) with propranolol and the hypothalamic-pituitary-adrenal axis (HPA) with mifepristone. Blockade of both stress axes robustly reduced infarct volumes, decreased infection rate, and increased long-term survival of 2D2 and C57BL/6J wild-type mice. Despite these protective effects, blockade of SIDS increased CNS antigen-specific Type1 T helper cell (Th1) responses in the brains of 2D2 mice 14 d after middle cerebral artery occlusion. One month after experimental stroke, 2D2 mice developed signs of polyradiculitis, which were diminished by SIDS blockade. Adoptive transfer of CD4+ T cells, isolated from 2D2 mice, into lymphocyte-deficient Rag-1KO mice did not reveal differences between SIDS blockade and vehicle treatment in functional long-term outcome after stroke. In conclusion, inhibiting SIDS by pharmacological blockade of bodys stress axes increases autoreactive CNS antigen-specific T-cell responses in the brain but does not worsen functional long-term outcome after experimental stroke, even in a mouse model where CNS antigen-specific autoreactive T-cell responses are boosted.

Effective treatment with intravenous immunoglobulins reduces autoreactive T-cell response in patients with CIDP

Objective To investigate changes in autoreactive T-cell responses against PMP-22 and P2 antigen as well as a T-cell memory repertoire in patients with chronic inflammatory demyelinating polyneuropathy (CIDP) induced by repeated intravenous immunoglobulin (IVIg) treatment. Methods In an observational trial, we prepared cryopreserved human peripheral blood monocytes from blood from 34 patients with CIDP (18 treatment naïve and 16 maintenance IVIg treatment) and from 14 healthy controls (non-immune neuropathy and healthy control). Treatment response was defined by clinical evaluation. The autoantigen-specific T-cell response was analysed by enzyme linked immunosorbent spot (ELISPOT) assay before IVIg start (baseline) and at follow-up. The T-cell memory subsets were analysed by using flow cytometric analysis. Results Myelin-derived P2-specific and PMP-22-specific IFN-γ producers were increased in IVIg responders compared with non-responders before treatment, which decreased by repeated IVIg infusion cycles. Treatment responders but not non-responders showed higher frequencies of CD4 T effector memory (TEM) and T central memory frequencies at baseline compared with maintenance IVIg treatment patients and controls. In addition, IVIg treatment was associated with a significant reduction in CD8 TEM at follow-up. Conclusions Our data demonstrate that immunomodulatory treatment with IVIgs on a long-term basis reduces the autoreactive T-cell response against PMP-22 and P2-antigens, which may be influenced by the altered maintenance of CD8 and CD4 effector/memory T-cell subsets towards a more anti-inflammatory immune status. Elevated PMP-22 and P2-specific T-cell responses may serve as predictors for treatment responsiveness to IVIgs warranting validation in larger studies.

STRoke Adverse Outcome is Associated with NoSocomial Infections (STRAWINSKI): Procalcitonin Ultrasensitive-Guided Antibacterial Therapy in Severe Ischaemic Stroke Patients – Rationale and Protocol for a Randomized Controlled Trial:

Rationale Stroke-associated pneumonia is one of the most common causes of poor outcome in stroke patients. Clinical signs and laboratory parameters of stroke-associated infections are often inconclusive. Biomarkers may help to identify stroke patients at high risk for pneumonia and to guide physicians in an early antibiotic treatment, thereby improving stroke outcome. Aim The aim of the present study is to investigate whether procalcitonin ultrasensitive-guided antibiotic treatment improves functional outcome after severe ischaemic stroke by early treatment of pneumonia. Design STRAWINSKI is an investigator-initiated, multicentre, randomized, controlled trial with blinded assessment of outcome comparing procalcitonin ultrasensitive-guided antibiotic treatment with standard care. Study 200 patients with ischaemic stroke in the middle cerebral artery territory and a score >9 on the National Institutes of Health Stroke Scale will be included and randomly assigned to two groups. One group will receive procalcitonin-based antibiotic therapy guidance; the other group will receive standard stroke unit care. Outcomes The primary endpoint is functional outcome at day 90 after stroke on the modified Rankin Scale, dichotomized as favourable (0–4) or unfavourable outcome (5,–6). Secondary endpoints are time to first event of death, rehospitalization, or recurrent stroke; death rate, infection rate, and days with fever up to day 7; length of hospital stay and hospital discharge disposition; shift analysis of the modified Rankin Scale; Barthel Index and days alive and out of hospital at day 90; use of antibiotics until day 90; and modified Rankin Scale, Barthel Index, and infarct volume at day 180.

Neuromyelitis optica in a patient with an early onset demyelinating episode: Clinical and autoantibody findings

Recent clinical and laboratory findings have substantially advanced our understanding of neuromyelitis optica (NMO) as a humorally mediated, autoimmune disorder. We report on a patient who suffered a first episode of transverse myelitis at the age of 6 months following diphtheria-pertussis-tetanus (DPT) vaccination which had therefore been considered suggestive of acute disseminated encephalomyelitis (ADEM). Fifteen years later, the further disease course revealed typical NMO meeting all diagnostic criteria. This development points to a broad clinical and temporal heterogeneity of NMO, with ADEM probably occurring in the context of a shared autoimmune diathesis. Despite therapy response following B-cell depletion by rituximab, positive NMO-IgG autoantibody status remained unchanged, whereas direct testing for anti-aquaporin-4 (AQP-4)-antibodies was negative throughout. Our findings challenge the pathogenic relevance of NMO-IgG and indicate a varying diagnostic value of testing for NMO-IgG and AQP-4-autoantibodies.

Circulating lymphocyte and T memory subsets in glucocorticosteroid versus IVIG treated patients with CIDP

The present study compared lymphocyte and T memory subsets in currently untreated patients with chronic inflammatory demyelinating polyneuropathy (CIDP) to glucocorticosteroid (GS) and intravenous immunoglobulin (IVIG) treated patients. Peripheral blood from 48 CIDP patients (21 untreated who were either treatment naïve or without treatment during the last 3 months, 17 IVIG and 10 GS treatment) and from 12 age-matched controls was evaluated using flow cytometric analysis. Our data demonstrate that long-term GS treatment is associated with reduced frequencies of total CD4+ T cells, CD4+ memory subsets and NK cells while long-term IVIG treatment is associated with alterations of the CD8+ memory compartment. Reduction of CD4+ naïve T cell counts may explain the observation that GS treatment induces prolonged clinical remission compared to IVIG treatment.