Differential compartmentalization of myeloid cell phenotypes and responses towards the CNS in Alzheimer's disease.

Myeloid cells are suggested as an important player in Alzheimer´s disease (AD). However, its continuum of phenotypic and functional changes across different body compartments and their use as a biomarker in AD remains elusive. Here, we perform multiple state-of-the-art analyses to phenotypically and metabolically characterize immune cells between peripheral blood (n = 117), cerebrospinal fluid (CSF, n = 117), choroid plexus (CP, n = 13) and brain parenchyma (n = 13). We find that CSF cells increase expression of markers involved in inflammation, phagocytosis, and metabolism. Changes in phenotype of myeloid cells from AD patients are more pronounced in CP and brain parenchyma and upon in vitro stimulation, suggesting that AD-myeloid cells are more vulnerable to environmental changes. Our findings underscore the importance of myeloid cells in AD and the detailed characterization across body compartments may serve as a resource for future studies focusing on the assessment of these cells as biomarkers in AD.

Immune Pathways in Etiology, Acute Phase, and Chronic Sequelae of Ischemic Stroke.

Inflammation and immune mechanisms are crucially involved in the pathophysiology of the development, acute damage cascades, and chronic course after ischemic stroke. Atherosclerosis is an inflammatory disease, and, in addition to classical risk factors, maladaptive immune mechanisms lead to an increased risk of stroke. Accordingly, individuals with signs of inflammation or corresponding biomarkers have an increased risk of stroke. Anti-inflammatory drugs, such as IL (interleukin)-1ß blockers, methotrexate, or colchicine, represent attractive treatment strategies to prevent vascular events and stroke. Lately, the COVID-19 pandemic shows a clear association between SARS-CoV2 infections and increased risk of cerebrovascular events. Furthermore, mechanisms of both innate and adaptive immune systems influence cerebral damage cascades after ischemic stroke. Neutrophils, monocytes, and microglia, as well as T and B lymphocytes each play complex interdependent roles that synergize to remove dead tissue but also can cause bystander injury to intact brain cells and generate maladaptive chronic inflammation. Chronic systemic inflammation and comorbid infections may unfavorably influence both outcome after stroke and recurrence risk for further stroke. In addition, stroke triggers specific immune depression, which in turn can promote infections. Recent research is now increasingly addressing the question of the extent to which immune mechanisms may influence long-term outcome after stroke and, in particular, cause specific complications such as poststroke dementia or even poststroke depression.

Immunodepression, Infections, and Functional Outcome in Ischemic Stroke.

Stroke remains one of the main causes of mortality and morbidity worldwide. Immediately after stroke, a neuroinflammatory process starts in the brain, triggering a systemic immunodepression mainly through excessive activation of the autonomous nervous system. Manifestations of immunodepression include lymphopenia but also dysfunctional innate and adaptive immune cells. The resulting impaired antibacterial defenses render patients with stroke susceptible to infections. In addition, other risk factors like stroke severity, dysphagia, impaired consciousness, mechanical ventilation, catheterization, and older age predispose stroke patients for infections. Most common infections are pneumonia and urinary tract infection, both occur in ≈10% of the patients. Especially pneumonia increases unfavorable outcome and mortality in patients with stroke; systemic effects like hypotension, fever, delay in rehabilitation are thought to play a crucial role. Experimental and clinical data suggest that systemic infections enhance autoreactive immune responses against brain antigens and thus negatively affect outcome but convincing evidence is lacking. Prevention of poststroke infections by preventive antibiotic therapy did not improve functional outcome after stroke. Immunomodulatory approaches counteracting immunodepression to prevent stroke-associated pneumonia need to account for neuroinflammation in the ischemic brain and avoid further tissue damage. Experimental studies discovered interesting targets, but these have not yet been investigated in patients with stroke. A better understanding of the pathobiology may help to develop optimized approaches of preventive antibiotic therapy or immunomodulation to effectively prevent stroke-associated pneumonia while improving long-term outcome after stroke. In this review, we aim to characterize epidemiology, risk factors, cause, diagnosis, clinical presentation, and potential treatment of poststroke immunosuppression and associated infections.

A Primeval Mechanism of Tolerance to Desiccation Based on Glycolic Acid Saves Neurons in Mammals from Ischemia by Reducing Intracellular Calcium-Mediated Excitotoxicity.

Stroke is the second leading cause of death and disability worldwide. Current treatments, such as pharmacological thrombolysis or mechanical thrombectomy, reopen occluded arteries but do not protect against ischemia-induced damage that occurs before reperfusion or neuronal damage induced by ischemia/reperfusion. It has been shown that disrupting the conversion of glyoxal to glycolic acid (GA) results in a decreased tolerance to anhydrobiosis in Caenorhabditis elegans dauer larva and that GA itself can rescue this phenotype. During the process of desiccation/rehydration, a metabolic stop/start similar to the one observed during ischemia/reperfusion occurs. In this study, the protective effect of GA is tested in different ischemia models, i.e., in commonly used stroke models in mice and swine. The results show that GA, given during reperfusion, strongly protects against ischemic damage and improves functional outcome. Evidence that GA exerts its effect by counteracting the glutamate-dependent increase in intracellular calcium during excitotoxicity is provided. These results suggest that GA treatment has the potential to reduce mortality and disability in stroke patients.

Efficacy and safety of intratracheal IFN-γ treatment to reverse stroke-induced susceptibility to pulmonary bacterial infections.

Stroke-induced immunosuppression contributes to the development of stroke-associated pneumonia (SAP). Experiments in mice demonstrated that apoptosis of IFN-γ producing cells and reduced IFN-γ secretion resulted in impaired immune responses and the development of pneumonia after middle cerebral artery occlusion (MCAo). In the present study, we investigated the efficacy of intratracheal IFN-γ treatment to prevent SAP and demonstrated that modest benefits on pulmonary cytokine response in IFN-γ treated stroke mice did not prevent spontaneously developing infections and even slightly reduced bacterial clearance of aspirated pneumococci. Our results suggest that pulmonary IFN-γ treatment is not an effective preventive measure for SAP.

CD4+ T cells promote delayed B cell responses in the ischemic brain after experimental stroke.

CD4+ T lymphocytes are key mediators of tissue damage after ischemic stroke. However, their infiltration kinetics and interactions with other immune cells in the delayed phase of ischemia remain elusive. We hypothesized that CD4+ T cells facilitate delayed autoreactive B cell responses in the brain, which have been previously linked to post-stroke cognitive impairment (PSCI). Therefore, we treated myelin oligodendrocyte glycoprotein T cell receptor transgenic 2D2 mice of both sexes with anti-CD4 antibody following 60-minute middle cerebral artery occlusion and assessed lymphocyte infiltration for up to 72 days. Anti-CD4-treatment eliminated CD4+ T cells from the circulation and ischemic brain for 28 days and inhibited B cell infiltration into the brain, particularly in animals with large infarcts. Absence of CD4+ T cells did not influence infarct maturation or survival. Once the CD4+ population recovered in the periphery, both CD4+ T and B lymphocytes entered the infarct site forming follicle-like structures. Additionally, we provide further evidence for PSCI that could be attenuated by CD4 depletion. Our findings demonstrate that CD4+ T cells are essential in delayed B cell infiltration into the ischemic brain after stroke. Importantly, lymphocyte infiltration after stroke is a long-lasting process. As CD4 depletion improved cognitive functions in an experimental set-up, these findings set the stage to elaborate more specific immune modulating therapies in treating PSCI.

Transfer RNA fragments replace microRNA regulators of the cholinergic poststroke immune blockade.

Stroke is a leading cause of death and disability. Recovery depends on a delicate balance between inflammatory responses and immune suppression, tipping the scale between brain protection and susceptibility to infection. Peripheral cholinergic blockade of immune reactions fine-tunes this immune response, but its molecular regulators are unknown. Here, we report a regulatory shift in small RNA types in patient blood sequenced 2 d after ischemic stroke, comprising massive decreases of microRNA levels and concomitant increases of transfer RNA fragments (tRFs) targeting cholinergic transcripts. Electrophoresis-based size-selection followed by qRT-PCR validated the top six up-regulated tRFs in a separate cohort of stroke patients, and independent datasets of small and long RNA sequencing pinpointed immune cell subsets pivotal to these responses, implicating CD14+ monocytes in the cholinergic inflammatory reflex. In-depth small RNA targeting analyses revealed the most-perturbed pathways following stroke and implied a structural dichotomy between microRNA and tRF target sets. Furthermore, lipopolysaccharide stimulation of murine RAW 264.7 cells and human CD14+ monocytes up-regulated the top six stroke-perturbed tRFs, and overexpression of stroke-inducible tRF-22-WE8SPOX52 using a single-stranded RNA mimic induced down-regulation of immune regulator Z-DNA binding protein 1. In summary, we identified a "changing of the guards" between small RNA types that may systemically affect homeostasis in poststroke immune responses, and pinpointed multiple affected pathways, which opens new venues for establishing therapeutics and biomarkers at the protein and RNA level.

Impact of Key Nicotinic AChR Subunits on Post-Stroke Pneumococcal Pneumonia.

Pneumonia is the most frequent severe medical complication after stroke. An overactivation of the cholinergic signaling after stroke contributes to immunosuppression and the development of spontaneous pneumonia caused by Gram-negative pathogens. The α7 nicotinic acetylcholine receptor (α7nAChR) has already been identified as an important mediator of the anti-inflammatory pathway after stroke. However, whether the α2, α5 and α9/10 nAChR expressed in the lung also play a role in suppression of pulmonary innate immunity after stroke is unknown. In the present study, we investigate the impact of various nAChRs on aspiration-induced pneumonia after stroke. Therefore, α2, α5, α7 and α9/10 nAChR knockout (KO) mice and wild type (WT) littermates were infected with Streptococcus pneumoniae (S. pneumoniae) three days after middle cerebral artery occlusion (MCAo). One day after infection pathogen clearance, cellularity in lung and spleen, cytokine secretion in bronchoalveolar lavage (BAL) and alveolar-capillary barrier were investigated. Here, we found that deficiency of various nAChRs does not contribute to an enhanced clearance of a Gram-positive pathogen causing post-stroke pneumonia in mice. In conclusion, these findings suggest that a single nAChR is not sufficient to mediate the impaired pulmonary defense against S. pneumoniae after experimental stroke.

Acute nicotine administration stimulates ciliary activity via α3ß4 nAChR in the mouse trachea.

Mucociliary clearance, the continuous removal of mucus-trapped particles by cilia-driven directed transport of the airway lining fluid, is the primary innate defense mechanism of the airways. It is potently activated by acetylcholine (ACh) addressing muscarinic receptors with a currently less defined role of nicotinic ACh receptors (nAChR). We here set out to determine their contribution in driving ciliary activity in an explanted mouse trachea preparation utilizing selected agonists and antagonists and nAChR-subunit deficient mice. Nicotine (100 µM) induced an increase in ciliary beat frequency, accompanied by a sharp, but not long lasting increase in particle transport speed (PTS) on the mucosal surface showing marked desensitization within the next 30 min. Nicotine-induced PTS acceleration was sensitive to the general nAChR inhibitors mecamylamine and d-tubocurarine as well as to the α3ß4-nAChR antagonist α-conotoxin AulB, but not to other antagonists primarily addressing α3ß2-nAChR or α4-, α7- and α9-containing nAChR. Agonists at α3ß*-nAChR (epibatidine, cytisine), but not cotinine mimicked the effect. Tracheas from mice with genetic deletion of nAChR subunits α5, α7, α9, α10, α9/10, and ß2 retained full PTS response to nicotine, whereas this was entirely lost in tracheas from mice lacking the ß4-subunit. Collectively, our data show that nicotinic stimulation of α3ß4-nAChR acutely increases PTS to the same extent as the established strong activator ATP. In view of the marked desensitization observed in the present setting, the physiological relevance of these receptors in adapting mucociliary clearance to rapidly changing endogenous or environmental stimuli remains open.

IgG stimulated ß2 adrenergic receptor activation is attenuated in patients with ME/CFS.

BACKGROUND: There is emerging evidence of a network of natural autoantibodies against GPCR which is dysregulated in various diseases. ß2 adrenergic and M3 and M4 cholinergic receptor (ß2 AdR and M3/4 mAChR) antibodies were found to be elevated in a subset of ME/CFS patients. METHODS: We comparatively analyzed the effects of polyclonal IgG on ß2 AdR signaling and immune cell function in vitro. 16 IgG fractions were isolated from serum of 5 ME/CFS patients with elevated (CFS AABhigh) and 5 with normal levels (CFS AABnorm) of ß2 AdR autoantibodies, and from 6 healthy controls (HC). The effect of each IgG on ß-arrestin recruitment and cAMP production in ß2 AdR and M3/4R reporter cell lines was studied. Further effect of each IgG on human monocyte cytokine production and on T cell proliferation in vitro was analyzed. In addition, studies on cytokine production in ß2 AdR wild type and knockout mice splenocytes incubated with IgG fractions were performed. RESULTS: We found that IgGs from HC could stimulate ß-arrestin recruitment and cAMP production in ß2 AdR reporter cell lines whereas IgGs from CFS AABhigh had no effect. The IgG-mediated activation of ß2 AdR was confirmed in ß2 AdR wt and ko mice. In accordance with previous studies IgG fractions from HC inhibited LPS-induced TNFα and stimulated LPS-induced IL-10 production of monocytes. Further IgG fractions from HC enhanced proliferation of T-cells stimulated with anti-CD3/CD28. IgG fractions from CFS AABhigh patients had no significant effect on both cytokine production and T cell proliferation, while IgGs from CFS AABnorm had an intermediate effect. We could also observe that IgG can modulate the signaling of ß2 AdR ligands isoprenline and propranolol. CONCLUSIONS: We provide evidence that IgG can activate ß2 AdR. The ß2 AdR activation by IgG is attenuated in ME/CFS patients. A dysregulation of ß2 AdR function could explain many symptoms of ME/CFS.

Immune Mediator Profile in Aqueous Humor Differs in Patients with Primary Acquired Ocular Toxoplasmosis and Recurrent Acute Ocular Toxoplasmosis.

PURPOSE: To compare the intraocular cytokine and chemokine profiles in patients with acute primary acquired ocular toxoplasmosis (pOT) or recurrent ocular toxoplasmosis (rOT) and to correlate them with their clinical characteristics. METHODS: Aqueous humor samples were collected from 62 consecutive patients (21 pOT, 30 rOT, and 11 noninfected controls) and analyzed by multiplex assay. Correlations were assessed between cytokine/chemokine levels, type of inflammatory response (Th1, Th2, and Th17), and clinical characteristics. In all OT patients, the clinical diagnosis of either pOT or rOT was confirmed by positive intraocular Goldmann/Witmer-Desmonts coefficient. Correlations were assessed between a preselected panel of immune mediators and the clinical characteristics of OT. RESULTS: In pOT patients, increased levels of IL-2, IFN-γ, TNF-α, IL-15, IL-4, IL-5, IL-9, IL-13, IL-17, IL-1Rα, IL-6, IL-1ß, and chemokines MIP-1α, MIP-1ß, IP-10, Eotaxin, IL-8, RANTES, PDGF-bb, GM-CSF, G-CSF, and MCP-1 were found in comparison to those in controls (p < 0.05). Patients with rOT showed elevated levels of IL-2, IFN-γ, TNF-α, IL-15, IL-4, IL-5, IL-9, IL-17, IL-1Rα, IL-6, IL-1ß, and chemokines MIP-1α, IP-10, Eotaxin, IL-8, RANTES, PDGF-bb, G-CSF, and MCP-1 compared to controls (p < 0.05). In addition, IL-7 (p = 0.028) differed between pOT and rOT; IL-9 (p = 0.054) and IL-13 (p = 0.051) showed a tendency of higher concentration in pOT than in rOT. A negative correlation was found between IL-7 (p = 0.017) as well as IL-9 (p = 0.008) and the number of recurrences. Cytokine ratios showed no difference between pOT and rOT, indicating a dominant Th1-type response in both infectious groups. Moreover, a positive correlation was detected between IL-7, VEGF, IL-13 and age at aqueous humor sampling (p < 0.05). CONCLUSIONS: This study for the first time shows subtle differences between the intraocular cytokine profiles in patients with either acute pOT or rOT.

Exploratory Investigation of Intestinal Function and Bacterial Translocation After Focal Cerebral Ischemia in the Mouse.

Background and Purpose: The gut communicates with the brain bidirectionally via neural, humoral and immune pathways. All these pathways are affected by acute brain lesions, such as stroke. Brain-gut communication may therefore impact on the overall outcome after CNS-injury. Until now, contradictory reports on intestinal function and translocation of gut bacteria after experimental stroke have been published. Accordingly, we aimed to specifically investigate the effects of transient focal cerebral ischemia on intestinal permeability, gut associated lymphoid tissue and bacterial translocation in an exploratory study using a well-characterized murine stroke model. Methods: After 60 min of middle cerebral artery occlusion (MCAO) we assessed intestinal morphology (time points after surgery day 0, 3, 5, 14, 21) and tight junction protein expression (occludin and claudin-1 at day 1 and 3) in 12-week-old male C57Bl/6J mice. Lactulose/mannitol/sucralose test was performed to assess intestinal permeability 24-72 h after surgery. To investigate the influence of cerebral ischemia on the local immune system of the gut, main immune cell populations in Peyer's patches (PP) were quantified by flow cytometry. Finally, we evaluated bacterial translocation to extraintestinal organs 24 and 72 h after MCAO by microbiological culture and fluorescence in situ hybridization targeting bacterial 16S rRNA. Results: Transient MCAO decreased claudin-1 expression in the ileum but not in the colon. Intestinal morphology (assessed by light microscopy) and permeability did not change measurably after MCAO. After MCAO, animals had significantly fewer B cells in PP compared to naïve mice. Conclusions: In a murine model of stroke, which leads to large brain infarctions in the middle cerebral artery territory, we did not find evidence for overt alterations neither in gut morphology, barrier proteins and permeability nor presence of intestinal bacterial translocation.

Immunomodulatory treatment with systemic GM-CSF augments pulmonary immune responses and improves neurological outcome after experimental stroke.

Stroke-induced immunodepression is an independent risk factor for stroke-associated pneumonia (SAP). Granulocyte-macrophage colony stimulating factor (GM-CSF) has neuroprotective properties in experimental stroke and been demonstrated to reverse immunodepression in sepsis patients. However, whether GM-CSF restores immune function after stroke preventing SAP and improving outcome is unknown. Here, we demonstrated that GM-CSF treatment improved peripheral and pulmonary leukocyte numbers, peripheral cytokine responses, lowered lung bacterial burden in the early course and improved long-term functional outcome after experimental stroke. These data suggest that GM-CSF is promising for stroke treatment since it not only acts neuroprotective in the ischemic brain but may also protect against detrimental post-stroke infections.

Depletion of Cultivatable Gut Microbiota by Broad-Spectrum Antibiotic Pretreatment Worsens Outcome After Murine Stroke.

BACKGROUND AND PURPOSE: Antibiotics disturbing microbiota are often used in treatment of poststroke infections. A bidirectional brain-gut microbiota axis was recently suggested as a modulator of nervous system diseases. We hypothesized that gut microbiota may be an important player in the course of stroke. METHODS: We investigated the outcome of focal cerebral ischemia in C57BL/6J mice after an 8-week decontamination with quintuple broad-spectrum antibiotic cocktail. These microbiota-depleted animals were subjected to 60 minutes middle cerebral artery occlusion or sham operation. Infarct volume was measured using magnetic resonance imaging, and mice were monitored clinically throughout the whole experiment. At the end point, tissues were preserved for further analysis, comprising histology and immunologic investigations using flow cytometry. RESULTS: We found significantly decreased survival in the middle cerebral artery occlusion microbiota-depleted mice when the antibiotic cocktail was stopped 3 days before surgery (compared with middle cerebral artery occlusion specific pathogen-free and sham-operated microbiota-depleted mice). Moreover, all microbiota-depleted animals in which antibiotic treatment was terminated developed severe acute colitis. This phenotype was rescued by continuous antibiotic treatment or colonization with specific pathogen-free microbiota before surgery. Further, infarct volumes on day one did not differ between any of the experimental groups. CONCLUSIONS: Conventional microbiota ensures intestinal protection in the mouse model of experimental stroke and prevents development of acute and severe colitis in microbiota-depleted mice not given antibiotic protection after cerebral ischemia. Our experiments raise the clinically important question as to whether microbial colonization or specific microbiota are crucial for stroke outcome.

Cholinergic Pathway Suppresses Pulmonary Innate Immunity Facilitating Pneumonia After Stroke.

BACKGROUND AND PURPOSE: Temporary immunosuppression has been identified as a major risk factor for the development of pneumonia after acute central nervous system injury. Although overactivation of the sympathetic nervous system was previously shown to mediate suppression of systemic cellular immune responses after stroke, the role of the parasympathetic cholinergic anti-inflammatory pathway in the antibacterial defense in lung remains largely elusive. METHODS: The middle cerebral artery occlusion model in mice was used to examine the influence of the parasympathetic nervous system on poststroke immunosuppression. We used heart rate variability measurement by telemetry, vagotomy, α7 nicotinic acetylcholine receptor-deficient mice, and parasympathomimetics (nicotine, PNU282987) to measure and modulate parasympathetic activity. RESULTS: Here, we demonstrate a rapidly increased parasympathetic activity in mice after experimental stroke. Inhibition of cholinergic signaling by either vagotomy or by using α7 nicotinic acetylcholine receptor-deficient mice reversed pulmonary immune hyporesponsiveness and prevented pneumonia after stroke. In vivo and ex vivo studies on the role of α7 nicotinic acetylcholine receptor on different lung cells using bone marrow chimeric mice and isolated primary cells indicated that not only macrophages but also alveolar epithelial cells are a major cellular target of cholinergic anti-inflammatory signaling in the lung. CONCLUSIONS: Thus, cholinergic pathways play a pivotal role in the development of pulmonary infections after acute central nervous system injury.

Miniaturized bronchoscopy enables unilateral investigation, application, and sampling in mice.

Lung diseases, including pneumonia and asthma, are among the most prevalent human disorders, and murine models have been established to investigate their pathobiology and develop novel treatment approaches. Whereas bronchoscopy is valuable for diagnostic and therapeutic procedures in patients, no equivalent for small rodents has been established. Here, we introduce a miniaturized video-bronchoscopy system offering new opportunities in experimental lung research. With an outer diameter of 0.75 mm, it is possible to advance the optics into the main bronchi of mice. An irrigation channel allows bronchoalveolar lavage and unilateral application of substances to one lung. Even a unilateral infection is possible, enabling researchers to use the contralateral lung as internal control.