Poststroke Lung Infection by Opportunistic Commensal Bacteria Is Not Mediated by Their Expansion in the Gut Microbiota.

BACKGROUND: Respiratory and urinary tract infections are frequent complications in patients with severe stroke. Stroke-associated infection is mainly due to opportunistic commensal bacteria of the microbiota that may translocate from the gut. We investigated the mechanisms underlying gut dysbiosis and poststroke infection. METHODS: Using a model of transient cerebral ischemia in mice, we explored the relationship between immunometabolic dysregulation, gut barrier dysfunction, gut microbial alterations, and bacterial colonization of organs, and we explored the effect of several drug treatments. RESULTS: Stroke-induced lymphocytopenia and widespread colonization of lung and other organs by opportunistic commensal bacteria. This effect correlated with reduced gut epithelial barrier resistance, and a proinflammatory sway in the gut illustrated by complement and nuclear factor-κB activation, reduced number of gut regulatory T cells, and a shift of gut lymphocytes to γδT cells and T helper 1/T helper 17 phenotypes. Stroke increased conjugated bile acids in the liver but decreased bile acids and short-chain fatty acids in the gut. Gut fermenting anaerobic bacteria decreased while opportunistic facultative anaerobes, notably Enterobacteriaceae, suffered an expansion. Anti-inflammatory treatment with a nuclear factor-κB inhibitor fully abrogated the Enterobacteriaceae overgrowth in the gut microbiota induced by stroke, whereas inhibitors of the neural or humoral arms of the stress response were ineffective at the doses used in this study. Conversely, the anti-inflammatory treatment did not prevent poststroke lung colonization by Enterobacteriaceae. CONCLUSIONS: Stroke perturbs homeostatic neuro-immuno-metabolic networks facilitating a bloom of opportunistic commensals in the gut microbiota. However, this bacterial expansion in the gut does not mediate poststroke infection.

Clemastine Induces an Impairment in Developmental Myelination.

Abnormalities in myelination are associated to behavioral and cognitive dysfunction in neurodevelopmental psychiatric disorders. Thus, therapies to promote or accelerate myelination could potentially ameliorate symptoms in autism. Clemastine, a histamine H1 antagonist with anticholinergic properties against muscarinic M1 receptor, is the most promising drug with promyelinating properties. Clemastine penetrates the blood brain barrier efficiently and promotes remyelination in different animal models of neurodegeneration including multiple sclerosis, ischemia and Alzheimer's disease. However, its role in myelination during development is unknown. We showed that clemastine treatment during development increased oligodendrocyte differentiation in both white and gray matter. However, despite the increase in the number of oligodendrocytes, conduction velocity of myelinated fibers of corpus callosum decreased in clemastine treated mice. Confocal and electron microscopy showed a reduction in the number of myelinated axons and nodes of Ranvier and a reduction of myelin thickness in corpus callosum. To understand the mechanisms leading to myelin formation impairment in the presence of an excess of myelinating oligodendrocytes, we focused on microglial cells that also express muscarinic M1 receptors. Importantly, the population of CD11c+ microglia cells, necessary for myelination, as well as the levels of insulin growth factor-1 decrease in clemastine-treated mice. Altogether, these data suggest that clemastine impact on myelin development is more complex than previously thought and could be dependent on microglia-oligodendrocyte crosstalk. Further studies are needed to clarify the role of microglia cells on developmental myelination.

Dendritic Cells and Microglia Have Non-redundant Functions in the Inflamed Brain with Protective Effects of Type 1 cDCs.

Brain CD11c+ cells share features with microglia and dendritic cells (DCs). Sterile inflammation increases brain CD11c+ cells, but their phenotype, origin, and functions remain largely unknown. We report that, after cerebral ischemia, microglia attract DCs to the inflamed brain, and astroglia produce Flt3 ligand, supporting development and expansion of CD11c+ cells. CD11c+ cells in the inflamed brain are a complex population derived from proliferating microglia and infiltrating DCs, including a major subset of OX40L+ conventional cDC2, and also cDC1, plasmacytoid, and monocyte-derived DCs. Despite sharing certain morphological features and markers, CD11c+ microglia and DCs display differential expression of pattern recognition receptors and chemokine receptors. DCs excel CD11c- and CD11c+ microglia in the capacity to present antigen through MHCI and MHCII. Of note, cDC1s protect from brain injury after ischemia. We thus reveal aspects of the dynamics and functions of brain DCs in the regulation of inflammation and immunity.

Antigen-Dependent T Cell Response to Neural Peptides After Human Ischemic Stroke.

Ischemic stroke causes brain tissue damage and may release central nervous system (CNS)-specific peptides to the periphery. Neural antigen presentation in the lymphoid tissue could prime immune cells and result in adaptive immune response. However, autoimmune responses against neural antigens are not commonly uncovered after stroke. We studied the brain tissue of nine fatal stroke cases and the blood of a cohort of 13 patients and 11 controls. Flow cytometry carried out in three of the brain samples showed CD8 and CD4 T cells in the cerebrospinal fluid (CSF) of the ventricles in the patient deceased 1 day poststroke, T cells with an activated phenotype in the CSF of the patient that died at day 6, and T cells in the ischemic brain tissue in the patient deceased 140 days after stroke onset. Immunohistochemistry showed higher T cell numbers in the core of the lesion of the patient deceased 18 days post-stroke than in the patients deceased from 1 to 5 days post-stroke. In blood samples, we studied whether lymphocytes were primed in the periphery against neural antigens at sequential times (on admission, day 5, and day 90) after stroke. T lymphocytes of stroke patients produced IFN-γ and TNF-α and responded to MBP peptides by increasing their production of TNF-α and IL-10 at admission, but not at later time points. In contrast, IL-4 producing T cells showed progressive increases. Higher percentages of TNF-α producing T lymphocytes at admission were independently associated with poorer outcomes at 90 days. However, we did not detect T cell responses to neural-antigen stimulation 90 days post-stroke. Altogether the results suggest acute T cell priming in the periphery in acute stroke, T cell trafficking from the CSF to the ischemic brain tissue, and the existence of active mechanisms preventing autoreactivity.

CCR2 deficiency in monocytes impairs angiogenesis and functional recovery after ischemic stroke in mice.

Inflammatory Ly6ChiCCR2+ monocytes infiltrate the brain after stroke but their functions are not entirely clear. We report that CCR2+ monocytes and CCR2+ lymphocytes infiltrate the brain after permanent ischemia. To underscore the role of CCR2+ monocytes, we generated mice with selective CCR2 deletion in monocytes. One day post-ischemia, these mice showed less infiltrating monocytes and reduced expression of pro-inflammatory cytokines, markers of alternatively macrophage activation, and angiogenesis. Accordingly, Ly6Chi monocytes sorted from the brain of wild type mice 24 h post-ischemia expressed pro-inflammatory genes, M2 genes, and pro-angiogenic genes. Flow cytometry showed heterogeneous phenotypes within the infiltrating Ly6ChiCCR2+ monocytes, including a subgroup of Arginase-1+ cells. Mice with CCR2-deficient monocytes displayed a delayed inflammatory rebound 15 days post-ischemia that was not found in wild type mice. Furthermore, they showed reduced angiogenesis and worse behavioral performance. Administration of CCR2+/+ bone-marrow monocytes to mice with CCR2-deficient monocytes did not improve the behavioral performance suggesting that immature bone-marrow monocytes lack pro-reparative functions. The results show that CCR2+ monocytes contribute to acute post-ischemic inflammation and participate in functional recovery. The study unravels heterogeneity in the population of CCR2+ monocytes infiltrating the ischemic brain and suggests that pro-reparative monocyte subsets promote functional recovery after ischemic stroke.

Microglial cell loss after ischemic stroke favors brain neutrophil accumulation.

Stroke attracts neutrophils to the injured brain tissue where they can damage the integrity of the blood-brain barrier and exacerbate the lesion. However, the mechanisms involved in neutrophil transmigration, location and accumulation in the ischemic brain are not fully elucidated. Neutrophils can reach the perivascular spaces of brain vessels after crossing the endothelial cell layer and endothelial basal lamina of post-capillary venules, or migrating from the leptomeninges following pial vessel extravasation and/or a suggested translocation from the skull bone marrow. Based on previous observations of microglia phagocytosing neutrophils recruited to the ischemic brain lesion, we hypothesized that microglial cells might control neutrophil accumulation in the injured brain. We studied a model of permanent occlusion of the middle cerebral artery in mice, including microglia- and neutrophil-reporter mice. Using various in vitro and in vivo strategies to impair microglial function or to eliminate microglia by targeting colony stimulating factor 1 receptor (CSF1R), this study demonstrates that microglial phagocytosis of neutrophils has fundamental consequences for the ischemic tissue. We found that reactive microglia engulf neutrophils at the periphery of the ischemic lesion, whereas local microglial cell loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils first in perivascular spaces and later in the parenchyma. Accordingly, microglia depletion by long-term treatment with a CSF1R inhibitor increased the numbers of neutrophils and enlarged the ischemic lesion. Hence, microglial phagocytic function sets a critical line of defense against the vascular and tissue damaging capacity of neutrophils in brain ischemia.