BackgroundUlcerative colitis (UC) is a chronic inflammatory disease marked by mucosal and systemic immune dysregulation. Fatigue is a common, burdensome extraintestinal symptom that often persists beyond active inflammation and affects quality of life. Sex-based differences in immune response and fatigue severity have been reported, but their mechanistic basis remain unclear. This study aimed to characterize peripheral immune profiles across UC disease stages and explore links between immunity, fatigue, and sex.MethodsEighty-nine individuals were enrolled: active UC (A; n=29), remission (R; n=30), and healthy controls (C; n=30). Flow cytometry assessed peripheral neutrophils, monocytes, dendritic cells (DCs), and T cell subsets, alongside plasma cytokines (BDNF, TNF, IL-6, IL-18, sTREM-2). Fatigue was evaluated using the validated Inflammatory Bowel Disease Fatigue (IBD-F) questionnaire, with sex-stratified correlation analyses.ResultsActive UC was associated with increased neutrophils and classical monocyte, alongside elevated TNF, BDNF, and sTREM-2. Upregulated CD62L expression and CCR2 expression in neutrophils and monocyte subsets indicated ongoing immune cell trafficking. During remission, increased plasmacytoid and CD141+ DCs, and Th9/Th22 cells suggested protective immune modulation. Fatigue severity correlated with specific immune subsets in a sex-dependent manner: in males, fatigue inversely correlated with classical monocytes and Th9/Th17/Tregs; in females, with NK cells and Tregs. Fatigue scores were higher in female patients.DiscussionPeripheral immune dysregulation in UC correlates with fatigue severity in a sex-specific manner. Our findings underscore the relevance of gut-brain axis signaling and highlight immune biomarkers with potential for stratified fatigue management in UC.

Ulcerative colitis (UC) patients experience cycles of active gut inflammation and remission, with neuropsychiatric comorbidities persisting even during clinical remission. While the dextran sulfate sodium (DSS) mouse model was previously applied to study gut-brain interactions, those studies focused on acute protocols missing the chronic, relapsing-remitting nature of human UC, when patients continue to experience central nervous system symptoms. Thus, we employed a chronic DSS treatment regimen comprising three cycles of active intestinal inflammation followed by remission phases to investigate region-specific microglial dynamics and their functional consequences on neuronal synapses. Transient blood-brain barrier alteration was detected during active chronic inflammation that was resolved during remission. Cortical microglia exhibited sustained iNOS-enriched activation state during remission, which coincided with synaptic imbalance: VGLUT1+ glutamatergic synaptosomes increased significantly in remission, while VGAT+ GABAergic vesicles declined, alongside suppressed neuronal c-fos expression. Hippocampal microglia adopted an ARG1-dominant phenotype with enriched TREM2, P2Y12R, and F4/80 expression during remission, indicating a phagocytic, reparative state. Hippocampal synaptosome analysis revealed selective excitatory enhancement with preserved inhibitory markers. Morphological analysis confirmed region-specific remodeling: cortical microglia displayed delayed process elaboration during remission, while hippocampal responses varied from transient (CA1) to persistent (CA3) somatic hypertrophy. These findings establish that remission from peripheral inflammation does not fully restore brain immune homeostasis. Persistent, region-specific microglial reactivity with cortical pro-inflammatory states associated with synaptic dysfunction and hippocampal adaptive responses preserving circuit integrity provides a mechanistic understanding for neuropsychiatric comorbidities in UC and suggests that brain-targeted therapies may be required to address the full disease burden.

Chronic infection with the protozoan parasite Toxoplasma gondii (T. gondii) elicits distinct alterations in both the immune and nervous system of the host. Previous studies correlated the persistent neuroinflammatory response triggered by chronic T. gondii infection to specific behavioral alterations. Here, we causally link chronic cerebral T. gondii infection to cognitive and motor impairments in mice, as well as to the altered brain glutamatergic signaling in hippocampus, striatum, and cortex. By combining synaptic composition analysis assessed via flow synaptometry with the standard sulfadiazine treatment, we demonstrated the regional specificity of the detected alterations of cerebral T. gondii infection. Importantly, our behavioral analysis exposed the restoration of behavioral flexibility in shifting between goal-directed and habitual action control, with more motorically demanding skills such as social novelty recognition and locomotion being only partially restored. We argue that the revealed regional effects of both T. gondii and sulfadiazine treatment may be a key factor accounting for treatment-resistant behavioral traits.

Background Chronic arterial hypertension causes cerebral microvascular dysfunction and increases dementia risk in aging. However, cognitive health preservation by therapeutic blood pressure lowering alone is limited and depends on disease duration, the degree of irreversible tissue damage, and whether microvascular function can be restored. This study aimed to understand molecular and cellular temporospatial mechanisms of disease in the course of hypertension. Methods We investigated the effects of initial, early chronic and late chronic hypertension in the frontal brain of spontaneously hypertensive stroke‐prone rats by applying behavioral tests, histopathology, immunofluorescence, fluorescence‐activated cell sorting, microvascular/neural tissue RNA sequencing, and 18 F‐fluorodeoxyglucose positron emission tomography imaging. Results Chronic hypertension caused behavioral deficits associated with frontal cortex function. Our results highlight stage‐dependent responses to continuous microvascular stress and wounding by hypertension. Early chronic responses included a fast recruitment of activated microglia to the blood vessels, immigration of peripheral immune cells, blood–brain barrier breakdown and an energy‐demanding hypermetabolic state. Vascular adaptation mechanisms were observed in later stages and included angiogenesis and upregulation of cellular adhesion molecules and extracellular matrix. Among the top upregulated genes in blood vessels, we identified Igfbp‐5 , which attenuates protective insulin‐like growth factor 1 signaling. Conclusions Our study provides new insight into mechanisms underlying hypertensive pathobiology and highlights its stage‐dependent nature. This groundwork will be helpful for basic and clinical research to identify stage‐dependent markers in the human disease course, investigate stage‐dependent interventions besides blood pressure lowering, and better understand the relationship between poor vascular health and neurodegenerative diseases.

IntroductionAging is accompanied by immunoscenescence and chronic low-grade inflammation (inflammaging), contributing to age-related diseases. Physical exercise is a potent modulator of immune function and systemic inflammation, yet the effects of acute exercise intensity on immune activation, cytokine dynamics, and extracellular vesicle release in older adults remain incompletely characterized, particularly in a sex-specific context. This study investigated how a single session of acute continuous moderate versus intense exercise modulates immune cell subsets, cytokine levels, and EV profiles in healthy older individuals, with emphasis on sex-based differences.MethodsThirty-three older adults completed either a moderate (n=14, 54-79 years; 60% VO2max, 30 minutes) or an intense cycling bout (n=19, 61-85 years; incremental cardiopulmonary exercise test (CPET) to exhaustion). Peripheral blood was collected at baseline, 30 minutes, and 24 hours post-exercise. Immune cells were analyzed by flow cytometry. EVs were characterized by flow cytometry and nanoparticle tracking analysis, and cytokines were quantified by multiplex assays.ResulsModerate exercise enhanced classical monocyte activation (↑CD86, ↓CX3CR1) without altering cell counts, and selectively elevated IL-6 in females. Intense exercise induced stronger innate immune activation, increasing classical and nonclassical monocytes, CD56bright/CD16low NK cells, and sustained TNFα levels. EVs positive for tetraspanins (CD9, CD63, and CD81) were elevated 24h after intense CPET. Exploratory sex-disaggregated analyses revealed distinct profiles: females had increased CD4+ EVs, while males showed elevated HLA-ABC+ EVs.DiscussionAcute exercise modulates immune responses in an intensity- and sex-dependent manner in older adults. Extracellular vesicle release was assessed only in the high-intensity intervention, where significant changes were observed. These findings support personalized exercise regimens to enhance immune resilience and promote healthy aging.

Chronic arterial hypertension restructures the vascular architecture of the brain, leading to a series of pathological responses that culminate in cerebral small vessel disease. Pericytes respond dynamically to vascular challenges; however, how they manifest under the continuous strain of hypertension has not been elucidated. Therefore, in this study, we characterized pericyte behavior alongside hypertensive states in the spontaneously hypertensive stroke-prone rat (SHRSP) model, emphasizing their phenotypic and metabolic transformation. Our results reveal an early transition in PDGFRß+ pericytes toward increased NG2 and CD13 co-expressing subtypes, signaling enhanced pericyte reactivity in an effort to stabilize vascular structures and an inflammatory engagement within the vascular niche in response to hypertensive stress. Gene expression profiling of microvessels revealed altered expression within crucial pathways i.e., angiogenesis, blood-brain barrier integrity, hypoxia and inflammation. Furthermore, we detected that circulating extracellular vesicles from SHRSP alter pericyte mitochondrial membrane potential, highlighting their ability to transmit pathogenic signals that exacerbate vascular remodeling. Detailed metabolic analysis revealed a significant shift toward glycolytic metabolism in pericytes already in initial hypertension, alongside a dysregulation of ATP production pathways. These findings emphasize the transformative influence of hypertension on cerebral pericytes and the extensive consequences on cerebral vascular health.### Competing Interest StatementThe authors have declared no competing interest.

Background Chronic arterial hypertension restructures the vascular architecture of the brain, leading to a series of pathological responses that culminate in cerebral small‐vessel disease. Pericytes respond dynamically to vascular challenges; however, how they manifest under the continuous strain of hypertension has not been elucidated. Methods and Results In this study, we characterized pericyte behavior alongside hypertensive states in the spontaneously hypertensive stroke‐prone rat model, focusing on their phenotypic and metabolic transformation. Flow cytometry was used to characterize pericytes by their expression of platelet‐derived growth factor receptor β, neuroglial antigen 2, cluster of differentiation 13–alanyl aminopeptidase, and antigen Kiel 67. Microvessels were isolated for gene expression profiling and in vitro pericyte expansion. Immunofluorescence validated the cell culture model. Plasma‐derived extracellular vesicles from hypertensive rodents were applied as a treatment to assess their effects on pericyte function and detailed metabolic assessments on enriched pericytes measured oxidative phosphorylation and glycolysis. Our results reveal a shift in platelet‐derived growth factor receptor β + pericytes toward increased neuroglial antigen 2 and cluster of differentiation 13–alanyl aminopeptidase coexpression, indicative of their critical role in vascular stabilization and inflammatory responses within the hypertensive milieu. Significant alterations were found within key pathways including angiogenesis, blood–brain barrier integrity, hypoxia, and inflammation. Circulating extracellular vesicles from hypertensive rodents distinctly influenced pericyte mitochondrial function, evidencing their dual role as carriers of disease pathology and potential therapeutic agents. Furthermore, a shift toward glycolytic metabolism in hypertensive pericytes was confirmed, coupled with ATP production dysregulation. Conclusions Our findings demonstrate that cerebral pericytes undergo phenotypic and metabolic reprogramming in response to hypertension, with hypertensive‐derived plasma‐derived extracellular vesicles impairing their mitochondrial function. Importantly, plasma‐derived extracellular vesicles from normotensive controls restore this function, suggesting their potential as both therapeutic agents and precision biomarkers for hypertensive vascular complications. Further investigation into plasma‐derived extracellular vesicle cargo is essential to further explore their therapeutic potential in vascular health.

Collagen type XVIII (COL18) is an abundant heparan sulfate proteoglycan in vascular basement membranes. Here, we asked (i) if the loss of COL18 would result in blood-brain barrier (BBB) breakdown, pathological alterations of small arteries and capillaries and neuroinflammation as found in cerebral small vessel disease (CSVD) and (ii) if such changes may be associated with remodeling of synapses and neural extracellular matrix (ECM). We found that 5-month-old Col18a1−/− mice had elevated BBB permeability for mouse IgG in the deep gray matter, and intravascular erythrocyte accumulations were observed brain-wide in capillaries and arterioles. BBB permeability increased with age and affected cortical regions and the hippocampus in 12-month-old Col18a1−/− mice. None of the Col18a1−/− mice displayed hallmarks of advanced CSVD, such as hemorrhages, and did not show perivascular space enlargement. Col18a1 deficiency-induced BBB leakage was accompanied by activation of microglia and astrocytes, a loss of aggrecan in the ECM of perineuronal nets associated with fast-spiking inhibitory interneurons and accumulation of the perisynaptic ECM proteoglycan brevican and the microglial complement protein C1q at excitatory synapses. As the pathway underlying these regulations, we found increased signaling through the TGF-ß1/Smad3/TIMP-3 cascade. We verified the pivotal role of COL18 for small vessel wall structure in CSVD by demonstrating the protein's involvement in vascular remodeling in autopsy brains from patients with cerebral hypertensive arteriopathy. Our study highlights an association between the alterations of perivascular ECM, extracellular proteolysis, and perineuronal/perisynaptic ECM, as a possible substrate of synaptic and cognitive alterations in CSVD.

Chronic arterial hypertension disrupts the integrity of the cerebral microvasculature, doubling the risk of age-related dementia. Despite sufficient antihypertensive therapy in still a significant proportion of individuals blood pressure lowering alone does not preserve cognitive health. Accumulating evidence highlights the role of inflammatory mechanisms in the pathogenesis of hypertension. In this review, we introduce a temporal framework to explore how early immune system activation and interactions at neurovascular-immune interfaces pave the way to cognitive impairment. The overall paradigm suggests that prohypertensive stimuli induce mechanical stress and systemic inflammatory responses that shift peripheral and meningeal immune effector mechanisms toward a proinflammatory state. Neurovascular-immune interfaces in the brain include a dysfunctional blood-brain barrier, crossed by peripheral immune cells; the perivascular space, in which macrophages respond to cerebrospinal fluid- and blood-derived immune regulators; and the meningeal immune reservoir, particularly T cells. Immune responses at these interfaces bridge peripheral and neurovascular unit inflammation, directly contributing to impaired brain perfusion, clearance of toxic metabolites, and synaptic function. We propose that deep immunophenotyping in biofluids together with advanced neuroimaging could aid in the translational determination of sequential immune and brain endotypes specific to arterial hypertension. This could close knowledge gaps on how and when immune system activation transits into neurovascular dysfunction and cognitive impairment. In the future, targeting specific immune mechanisms could prevent and halt hypertension disease progression before clinical symptoms arise, addressing the need for new interventions against one of the leading threats to cognitive health.

Extracellular vesicles (EVs) are key in intercellular communication, carrying biomolecules like nucleic acids, lipids, and proteins. This study investigated postprandial characteristics and proteomic profiles of blood-derived EVs in healthy individuals. Twelve participants fasted overnight before baseline assessments. After consuming a controlled isocaloric meal, EVs were isolated for proteomic and flow cytometric analysis. Plasma triacylglyceride levels confirmed fasting completion, while protein concentrations in plasma and EVs were monitored for postprandial stability. Proteomic analysis identified upregulated proteins related to transport mechanisms and epithelial/endothelial functions postprandially, indicating potential roles in physiological responses to nutritional intake. Enrichment analyses revealed vesicle-related pathways and immune system processes. Flow cytometry showed increased expression of CD324 on CD9+CD63+CD81+ large extracellular vesicles postprandially, suggesting an epithelial origin. These findings offer valuable insights into postprandial EV dynamics and their potential physiological significance, highlighting the need for stringent fasting guidelines in EV studies to account for postprandial effects on EV composition and function.

In a great partnership, the Federation of European Neuroscience Societies (FENS) and the Hertie Foundation organized the FENS-Hertie 2022 Winter School on 'Neuro-immune interactions in health and disease'. The school selected 27 PhD students and 13 postdoctoral fellows from 20 countries and involved 14 faculty members experts in the field. The Winter School focused on a rising field of research, the interactions between the nervous and both innate and adaptive immune systems under pathological and physiological conditions. A fine-tuned neuro-immune crosstalk is fundamental for healthy development, while disrupted neuro-immune communication might play a role in neurodegeneration, neuroinflammation and aging. However, much is yet to be understood about the underlying mechanisms of these neuro-immune interactions in the healthy brain and under pathological scenarios. In addition to new findings in this emerging field, novel methodologies and animal models were presented to foment research on neuro-immunology. The FENS-Hertie 2022 Winter School provided an insightful knowledge exchange between students and faculty focusing on the latest discoveries in the biology of neuro-immune interactions while fostering great academic and professional opportunities for early-career neuroscientists from around the world.

Regular physical activity is a cornerstone of healthy aging, offering a wide range of benefits, including the modulation of immune regulation and reduction of chronic inflammation. With aging closely linked to persistent, low-grade inflammation, i.e. inflammaging, the effects of exercise intensity on acute immune responses in older adults remain not fully understood. In this study, we explored how moderate and intense acute continuous exercise impact immune cell activation, cytokine production and large extracellular vesicle (lEV) release in healthy elderly individuals. Fourteen participants completed a moderate continuous exercise intervention (60% VO2max for 30 minutes), while nineteen engaged in an intense continuous exercise session until exhaustion. Blood samples were collected at baseline, and at 1- and 24-hours post-exercise. Immune cell characterization by flow cytometry revealed distinct changes in monocyte subsets and NK cells activation across both exercise intensities. Intense exercise was associated with elevated proinflammatory TNFα levels, accumulation of circulating plasma-derived lEV and changes in their surface marker expression after 24 hours. Additionally, we identified sex-specific differences, including distinct activation profiles in innate immunity, alterations in EV release from CD4+ and HLA+ cells, and an exercise-induced increase in IL-6 observed exclusively in females. These findings suggest that moderate continuous acute exercise enhances immune cell activation without altering cell counts, while intense continuous exercise triggers acute proinflammatory immune response. Further research should clarify the long-term implications and fundamental mechanisms of exercise-induced immune modulation in aging populations. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the following grants: German Center for Mental Health (DZPG) (funded by BMBF (to PM & ID), European Regional Development Fund (EFRE) (ZS/2024/02/184014, to RBD, SS, ID & PM), Graduate scholarship from the Novartis Foundation (to PM), Polycarp-Leporin-Program (PLP23/5,to PM). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study protocol was approved by the Ethics Committee of the Medical Faculty at Otto-von-Guericke University in Magdeburg, Germany (reference number: 07/20). All the procedures were conducted in accordance with the ethical standards outlined in the Declaration of Helsinki. Prior to participation, each individual provided written informed consent following a comprehensive explanation of the study, and all queries were addressed satisfactorily. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Extracellular vesicles (EVs) are key in intercellular communication, carrying biomolecules like nucleic acids, lipids, and proteins. This study investigated postprandial characteristics and proteomic profiles of circulating large extracellular vesicles (lEVs) in healthy individuals. Twelve participants fasted overnight before baseline assessments. After consuming a controlled isocaloric meal, lEVs were isolated for proteomic and flow cytometric analysis. Plasma triacylglyceride (TAG) levels confirmed fasting completion, while protein concentrations in plasma and lEVs were monitored for postprandial stability. Proteomic analysis identified upregulated proteins related to transport mechanisms and epithelial/endothelial functions postprandially, indicating potential roles in physiological responses to nutritional intake. Enrichment analyses revealed vesicle-related pathways and immune system processes. Flow cytometry showed increased expression of CD324 on medium-sized CD9+CD63+CD81+ EVs postprandially, suggesting an epithelial origin. These findings offer insights into postprandial lEV dynamics and their physiological significance, highlighting the need for stringent fasting guidelines in EV studies to account for postprandial effects on EV composition and function.

Brain vascular health appears to be critical for preventing the development of amyotrophic lateral sclerosis (ALS) and slowing its progression. ALS patients often demonstrate cardiovascular risk factors and commonly suffer from cerebrovascular disease, with evidence of pathological alterations in their small cerebral blood vessels. Impaired vascular brain health has detrimental effects on motor neurons: vascular endothelial growth factor levels are lowered in ALS, which can compromise endothelial cell formation and the integrity of the blood–brain barrier. Increased turnover of neurovascular unit cells precedes their senescence, which, together with pericyte alterations, further fosters the failure of toxic metabolite removal. We here provide a comprehensive overview of the pathogenesis of impaired brain vascular health in ALS and how novel magnetic resonance imaging techniques can aid its detection. In particular, we discuss vascular patterns of blood supply to the motor cortex with the number of branches from the anterior and middle cerebral arteries acting as a novel marker of resistance and resilience against downstream effects of vascular risk and events in ALS. We outline how certain interventions adapted to patient needs and capabilities have the potential to mechanistically target the brain microvasculature towards favorable motor cortex blood supply patterns. Through this strategy, we aim to guide novel approaches to ALS management and a better understanding of ALS pathophysiology.

Tobacco smoking is strongly linked to vascular damage contributing to the development of hypertension, atherosclerosis, as well as increasing the risk for neurodegeneration. Still, the involvement of the innate immune system in the development of vascular damage upon chronic tobacco use before the onset of clinical symptoms is not fully characterized. Our data provide evidence that a single acute exposure to tobacco elicits the secretion of extracellular vesicles expressing CD105 and CD49e from endothelial cells, granting further recognition of early preclinical biomarkers of vascular damage. Furthermore, we investigated the effects of smoking on the immune system of healthy asymptomatic chronic smokers compared to never-smokers, focusing on the innate immune system. Our data reveal a distinct immune landscape representative for early stages of vascular damage in clinically asymptomatic chronic smokers, before tobacco smoking related diseases develop. These results indicate a dysregulated immuno-vascular axis in chronic tobacco smokers that are otherwise considered as healthy individuals. The distinct alterations are characterized by increased CD36 expression by the blood monocyte subsets, neutrophilia and increased plasma IL-18 and reduced levels of IL-33, IL-10 and IL-8. Additionally, reduced levels of circulating BDNF and elevated sTREM2, which are associated with neurodegeneration, suggest a considerable impact of tobacco smoking on CNS function in clinically healthy individuals. These findings provide profound insight into the initial and ongoing effects of tobacco smoking and the potential vascular damage contributing to neurodegenerative disorders, specifically cerebrovascular dysfunction and dementia.

Vascular risk factors such as chronic hypertension are well-established major modifiable factors for the development of cerebral small vessel disease (cSVD). In the present study, our focus was the investigation of cSVD-related phenotypic changes in microglia in human disease and in the spontaneously hypertensive stroke-prone rat (SHRSP) model of cSVD. Our examination of cortical microglia in human post-mortem cSVD cortical tissue revealed distinct morphological microglial features specific to cSVD. We identified enlarged somata, an increase in the territory occupied by thickened microglial processes, and an expansion in the number of vascular-associated microglia. In parallel, we characterized microglia in a rodent model of hypertensive cSVD along different durations of arterial hypertension, i.e., early chronic and late chronic hypertension. Microglial somata were already enlarged in early hypertension. In contrast, at late-stage chronic hypertension, they further exhibited elongated branches, thickened processes, and a reduced ramification index, mirroring the findings in human cSVD. An unbiased multidimensional flow cytometric analysis revealed phenotypic heterogeneity among microglia cells within the hippocampus and cortex. At early-stage hypertension, hippocampal microglia exhibited upregulated CD11b/c, P2Y12R, CD200R, and CD86 surface expression. Detailed analysis of cell subpopulations revealed a unique microglial subset expressing CD11b/c, CD163, and CD86 exclusively in early hypertension. Notably, even at early-stage hypertension, microglia displayed a higher association with cerebral blood vessels. We identified several profound clusters of microglia expressing distinct marker profiles at late chronic hypertensive states. In summary, our findings demonstrate a higher vulnerability of the hippocampus, stage-specific microglial signatures based on morphological features, and cell surface protein expression in response to chronic arterial hypertension. These results indicate the diversity within microglia sub-populations and implicate the subtle involvement of microglia in cSVD pathogenesis.

Abstract Background Toxoplasma gondii (T. gondii) is a highly successful parasite being able to cross all biological barriers of the body, finally reaching the central nervous system (CNS). Previous studies have highlighted the critical involvement of the blood–brain barrier (BBB) during T. gondii invasion and development of subsequent neuroinflammation. Still, the potential contribution of the choroid plexus (CP), the main structure forming the blood–cerebrospinal fluid (CSF) barrier (BCSFB) have not been addressed. Methods To investigate T. gondii invasion at the onset of neuroinflammation, the CP and brain microvessels (BMV) were isolated and analyzed for parasite burden. Additionally, immuno-stained brain sections and three-dimensional whole mount preparations were evaluated for parasite localization and morphological alterations. Activation of choroidal and brain endothelial cells were characterized by flow cytometry. To evaluate the impact of early immune responses on CP and BMV, expression levels of inflammatory mediators, tight junctions (TJ) and matrix metalloproteinases (MMPs) were quantified. Additionally, FITC-dextran was applied to determine infection-related changes in BCSFB permeability. Finally, the response of primary CP epithelial cells to T. gondii parasites was tested in vitro. Results Here we revealed that endothelial cells in the CP are initially infected by T. gondii, and become activated prior to BBB endothelial cells indicated by MHCII upregulation. Additionally, CP elicited early local immune response with upregulation of IFN-γ, TNF, IL-6, host-defence factors as well as swift expression of CXCL9 chemokine, when compared to the BMV. Consequently, we uncovered distinct TJ disturbances of claudins, associated with upregulation of MMP-8 and MMP-13 expression in infected CP in vivo, which was confirmed by in vitro infection of primary CP epithelial cells. Notably, we detected early barrier damage and functional loss by increased BCSFB permeability to FITC-dextran in vivo, which was extended over the infection course. Conclusions Altogether, our data reveal a close interaction between T. gondii infection at the CP and the impairment of the BCSFB function indicating that infection-related neuroinflammation is initiated in the CP.

Our results point towards stage-dependent reactions of vascular, glial, immune and neuronal cells in response to hypertension. Future studies have to verify, whether these mechanisms are valid in humans as well and whether they might be targeted to attenuate small vessel disease progression.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrated that MC-derived tumor necrosis factor (TNF) was crucial for neutrophil extravasation to sites of contact hypersensitivity-induced skin inflammation by promoting intraluminal crawling. MC-derived TNF directly primed circulating neutrophils via TNF receptor-1 (TNFR1) while being dispensable for endothelial cell activation. The MC-derived TNF was infused into the bloodstream by directional degranulation of perivascular MCs that were part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules boosted neutrophil extravasation. Pronounced and rapid intravascular MC degranulation was also observed upon IgE crosslinking or LPs challenge indicating a universal MC potential. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.

Tissue resident mast cells (MCs) rapidly initiate neutrophil infiltration upon inflammatory insult, yet the molecular mechanism is still unknown. Here, we demonstrate that MC-derived TNF is crucial for neutrophil extravasation to sites of sterile skin inflammation by promoting intraluminal adhesion and crawling. Strikingly, MC-TNF directly primes circulating neutrophils via TNFR1, while being dispensable for chemokine production and endothelial cell activation. The MC-TNF is fused into the bloodstream by directional degranulation of perivascular MCs that are part of the vascular unit with access to the vessel lumen. Consistently, intravenous administration of MC granules can boost neutrophil extravasation. Consequently, the directional MC degranulation of pro-inflammatory mediators into the bloodstream may represent an important target for therapeutic approaches aimed at dampening cytokine storm syndromes or shock symptoms, or intentionally pushing immune defense.