Microvascular damage, neuroinflammation and extracellular matrix remodeling in Col18a1 knockout mice as a model for early cerebral small vessel disease.

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.

Astrocytes evoke a robust IRF7-independent type I interferon response upon neurotropic viral infection.

BACKGROUND: Type I interferons (IFN-I) are fundamental in controlling viral infections but fatal interferonopathy is restricted in the immune-privileged central nervous system (CNS). In contrast to the well-established role of Interferon Regulatory Factor 7 (IRF7) in the regulation of IFN-I response in the periphery, little is known about the specific function in the CNS. METHODS: To investigate the role for IRF7 in antiviral response during neurotropic virus infection, mice deficient for IRF3 and IRF7 were infected systemically with Langat virus (LGTV). Viral burden and IFN-I response was analyzed in the periphery and the CNS by focus formation assay, RT-PCR, immunohistochemistry and in vivo imaging. Microglia and infiltration of CNS-infiltration of immune cells were characterized by flow cytometry. RESULTS: Here, we demonstrate that during infection with the neurotropic Langat virus (LGTV), an attenuated member of the tick-borne encephalitis virus (TBEV) subgroup, neurons do not rely on IRF7 for cell-intrinsic antiviral resistance and IFN-I induction. An increased viral replication in IRF7-deficient mice suggests an indirect antiviral mechanism. Astrocytes rely on IRF7 to establish a cell-autonomous antiviral response. Notably, the loss of IRF7 particularly in astrocytes resulted in a high IFN-I production. Sustained production of IFN-I in astrocytes is independent of an IRF7-mediated positive feedback loop. CONCLUSION: IFN-I induction in the CNS is profoundly regulated in a cell type-specific fashion.

Phytohormones regulate asexual Toxoplasma gondii replication.

The protozoan Toxoplasma gondii (T. gondii) is a zoonotic disease agent causing systemic infection in warm-blooded intermediate hosts including humans. During the acute infection, the parasite infects host cells and multiplies intracellularly in the asexual tachyzoite stage. In this stage of the life cycle, invasion, multiplication, and egress are the most critical events in parasite replication. T. gondii features diverse cell organelles to support these processes, including the apicoplast, an endosymbiont-derived vestigial plastid originating from an alga ancestor. Previous studies have highlighted that phytohormones can modify the calcium-mediated secretion, e.g., of adhesins involved in parasite movement and cell invasion processes. The present study aimed to elucidate the influence of different plant hormones on the replication of asexual tachyzoites in a human foreskin fibroblast (HFF) host cell culture. T. gondii replication was measured by the determination of T. gondii DNA copies via qPCR. Three selected phytohormones, namely abscisic acid (ABA), gibberellic acid (GIBB), and kinetin (KIN) as representatives of different plant hormone groups were tested. Moreover, the influence of typical cell culture media components on the phytohormone effects was assessed. Our results indicate that ABA is able to induce a significant increase of T. gondii DNA copies in a typical supplemented cell culture medium when applied in concentrations of 20 ng/µl or 2 ng/µl, respectively. In contrast, depending on the culture medium composition, GIBB may potentially serve as T. gondii growth inhibitor and may be further investigated as a potential treatment for toxoplasmosis.

Classifying flow cytometry data using Bayesian analysis helps to distinguish ALS patients from healthy controls.

Introduction: Given its wide availability and cost-effectiveness, multidimensional flow cytometry (mFC) became a core method in the field of immunology allowing for the analysis of a broad range of individual cells providing insights into cell subset composition, cellular behavior, and cell-to-cell interactions. Formerly, the analysis of mFC data solely relied on manual gating strategies. With the advent of novel computational approaches, (semi-)automated gating strategies and analysis tools complemented manual approaches. Methods: Using Bayesian network analysis, we developed a mathematical model for the dependencies of different obtained mFC markers. The algorithm creates a Bayesian network that is a HC tree when including raw, ungated mFC data of a randomly selected healthy control cohort (HC). The HC tree is used to classify whether the observed marker distribution (either patients with amyotrophic lateral sclerosis (ALS) or HC) is predicted. The relative number of cells where the probability q is equal to zero is calculated reflecting the similarity in the marker distribution between a randomly chosen mFC file (ALS or HC) and the HC tree. Results: Including peripheral blood mFC data from 68 ALS and 35 HC, the algorithm could correctly identify 64/68 ALS cases. Tuning of parameters revealed that the combination of 7 markers, 200 bins, and 20 patients achieved the highest AUC on a significance level of p < 0.0001. The markers CD4 and CD38 showed the highest zero probability. We successfully validated our approach by including a second, independent ALS and HC cohort (55 ALS and 30 HC). In this case, all ALS were correctly identified and side scatter and CD20 yielded the highest zero probability. Finally, both datasets were analyzed by the commercially available algorithm 'Citrus', which indicated superior ability of Bayesian network analysis when including raw, ungated mFC data. Discussion: Bayesian network analysis might present a novel approach for classifying mFC data, which does not rely on reduction techniques, thus, allowing to retain information on the entire dataset. Future studies will have to assess the performance when discriminating clinically relevant differential diagnoses to evaluate the complementary diagnostic benefit of Bayesian network analysis to the clinical routine workup.

Deletion of p75NTR rescues the synaptic but not the inflammatory status in the brain of a mouse model for Alzheimer's disease.

Introduction: Alzheimer's disease (AD), is characterized by a gradual cognitive decline associated with the accumulation of Amyloid beta (Aß)-oligomers, progressive neuronal degeneration and chronic neuroinflammation. Among the receptors shown to bind and possibly transduce the toxic effects of Aß-oligomers is the p75 neurotrophin receptor (p75NTR). Interestingly, p75NTR mediates several crucial processes in the nervous system, including neuronal survival and apoptosis, maintenance of the neuronal architecture, and plasticity. Furthermore, p75NTR is also expressed in microglia, the resident immune cells of the brain, where it is markedly increased under pathological conditions. These observations indicate p75NTR as a potential candidate for mediating Aß-induced toxic effects at the interface between the nervous and the immune system, thereby potentially participating in the crosstalk between these two systems. Methods: Here we used APP/PS1 transgenic mice (APP/PS1tg) and compared the Aß-induced alterations in neuronal function, chronic inflammation as well as their cognitive consequences between 10 months old APP/PS1tg and APP/PS1tg x p75NTRexonIV knockout mice. Results: Electrophysiological recordings show that a loss of p75NTR rescues the impairment in long-term potentiation at the Schaffer collaterals in the hippocampus of APP/PS1tg mice. Interestingly, however loss of p75NTR does not influence the severity of neuroinflammation, microglia activation or the decline in spatial learning and memory processes observed in APP/PS1tg mice. Conclusion: Together these results indicate that while a deletion of p75NTR rescues the synaptic defect and the impairment in synaptic plasticity, it does not affect the progression of the neuroinflammation and the cognitive decline in a mouse model for AD.

Plasma concentrations of anti-inflammatory cytokine TGF-ß are associated with hippocampal structure related to explicit memory performance in older adults.

Human cognitive abilities, and particularly hippocampus-dependent memory performance typically decline with increasing age. Immunosenescence, the age-related disintegration of the immune system, is increasingly coming into the focus of research as a considerable factor contributing to cognitive decline. In the present study, we investigated potential associations between plasma levels of pro- and anti-inflammatory cytokines and learning and memory performance as well as hippocampal anatomy in young and older adults. Plasma concentrations of the inflammation marker CRP as well as the pro-inflammatory cytokines IL-6 and TNF-α and the anti-inflammatory cytokine TGF-ß1 were measured in 142 healthy adults (57 young, 24.47 ± 4.48 years; 85 older, 63.66 ± 7.32 years) who performed tests of explicit memory (Verbal Learning and Memory Test, VLMT; Wechsler Memory Scale, Logical Memory, WMS) with an additional delayed recall test after 24 h. Hippocampal volumetry and hippocampal subfield segmentation were performed using FreeSurfer, based on T1-weighted and high-resolution T2-weighted MR images. When investigating the relationship between memory performance, hippocampal structure, and plasma cytokine levels, we found that TGF-ß1 concentrations were positively correlated with the volumes of the hippocampal CA4-dentate gyrus region in older adults. These volumes were in turn positively associated with better performance in the WMS, particularly in the delayed memory test. Our results support the notion that endogenous anti-inflammatory mechanisms may act as protective factors in neurocognitive aging.

Brain Vascular Health in ALS Is Mediated through Motor Cortex Microvascular Integrity.

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.

Initial and ongoing tobacco smoking elicits vascular damage and distinct inflammatory response linked to neurodegeneration.

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.

The neuropeptide PACAP alleviates T. gondii infection-induced neuroinflammation and neuronal impairment.

BACKGROUND: Cerebral infection with the protozoan Toxoplasma gondii (T. gondii) is responsible for inflammation of the central nervous system (CNS) contributing to subtle neuronal alterations. Albeit essential for brain parasite control, continuous microglia activation and recruitment of peripheral immune cells entail distinct neuronal impairment upon infection-induced neuroinflammation. PACAP is an endogenous neuropeptide known to inhibit inflammation and promote neuronal survival. Since PACAP is actively transported into the CNS, we aimed to assess the impact of PACAP on the T. gondii-induced neuroinflammation and subsequent effects on neuronal homeostasis. METHODS: Exogenous PACAP was administered intraperitoneally in the chronic stage of T. gondii infection, and brains were isolated for histopathological analysis and determination of pathogen levels. Immune cells from the brain, blood, and spleen were analyzed by flow cytometry, and the further production of inflammatory mediators was investigated by intracellular protein staining as well as expression levels by RT-qPCR. Neuronal and synaptic alterations were assessed on the transcriptional and protein level, focusing on neurotrophins, neurotrophin-receptors and signature synaptic markers. RESULTS: Here, we reveal that PACAP administration reduced the inflammatory foci and the number of apoptotic cells in the brain parenchyma and restrained the activation of microglia and recruitment of monocytes. The neuropeptide reduced the expression of inflammatory mediators such as IFN-γ, IL-6, iNOS, and IL-1ß. Moreover, PACAP diminished IFN-γ production by recruited CD4+ T cells in the CNS. Importantly, PACAP promoted neuronal health via increased expression of the neurotrophin BDNF and reduction of p75NTR, a receptor related to neuronal cell death. In addition, PACAP administration was associated with increased expression of transporters involved in glutamatergic and GABAergic signaling that are particularly affected during cerebral toxoplasmosis. CONCLUSIONS: Together, our findings unravel the beneficial effects of exogenous PACAP treatment upon infection-induced neuroinflammation, highlighting the potential implication of neuropeptides to promote neuronal survival and minimize synaptic prejudice.

IFN-γ-mediated neuronal defense mechanism targets Toxoplasma.

Toxoplasma gondii encysts preferentially within neurons in the central nervous system, establishing lifelong persistence. Despite recent discoveries, this neuronal preference was thought, in part, to be secondary to a lack of neuronal cell-autonomous immunity. By showing that neurons can mount interferon-gamma (IFN-γ)-mediated cell-autonomous anti-T. gondii defenses, Chandrasekaran et al. have challenged long held assumptions.

Persisting Microbiota and Neuronal Imbalance Following T. gondii Infection Reliant on the Infection Route.

Toxoplasma gondii is a highly successful parasite capable of infecting all warm-blooded animals. The natural way of infection in intermediate hosts is the oral ingestion of parasite-contaminated water or food. In murine experimental models, oral infection (p.o.) of mice with T. gondii is applied to investigate mucosal and peripheral immune cell dynamics, whereas intraperitoneal infection (i.p.) is frequently used to study peripheral inflammation as well as immune cell - neuronal interaction in the central nervous system (CNS). However, the two infection routes have not yet been systematically compared along the course of infection. Here, C57BL/6 mice were infected p.o. or i.p. with a low dose of T. gondii cysts, and the acute and chronic stages of infection were compared. A more severe course of infection was detected following i.p. challenge, characterized by an increased weight loss and marked expression of proinflammatory cytokines particularly in the CNS during the chronic stage. The elevated proinflammatory cytokine expression in the ileum was more prominent after p.o. challenge that continued following the acute phase in both i.p. or p.o. infected mice. This resulted in sustained microbial dysbiosis, especially after p.o. challenge, highlighted by increased abundance of pathobionts from the phyla proteobacteria and a reduction of beneficial commensal species. Further, we revealed that in the CNS of i.p. infected mice CD4 and CD8 T cells displayed higher IFNγ production in the chronic stage. This corresponded with an increased expression of C1q and CD68 in the CNS and reduced expression of genes involved in neuronal signal transmission. Neuroinflammation-associated synaptic alterations, especially PSD-95, VGLUT, and EAAT2 expression, were more pronounced in the cortex upon i.p. infection highlighting the profound interplay between peripheral inflammation and CNS homeostasis.

Type 1 innate lymphoid cells regulate the onset of Toxoplasma gondii-induced neuroinflammation.

Cerebral infections are restrained by a complex interplay of tissue-resident and recruited peripheral immune cells. Whether innate lymphoid cells (ILCs) are involved in the orchestration of the neuroinflammatory dynamics is not fully understood. Here, we demonstrate that ILCs accumulate in the cerebral parenchyma, the choroid plexus, and the meninges in the onset of cerebral Toxoplasma gondii infection. Antibody-mediated depletion of conventional natural killer (cNK) cells and ILC1s in the early stage of infection results in diminished cytokine and chemokine expression and increased cerebral parasite burden. Using cNK- and ILC1-deficient murine models, we demonstrate that exclusively the lack of ILC1s affects cerebral immune responses. In summary, our results provide evidence that ILC1s are an early source of IFN-γ and TNF in response to cerebral T. gondii infection, thereby inducing host defense factors and initiating the development of a neuroinflammatory response.

Immune response and pathogen invasion at the choroid plexus in the onset of cerebral toxoplasmosis.

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.

Acute alcohol consumption increases systemic endotoxin bioactivity for days in healthy volunteers-with reduced intestinal barrier loss in female.

OBJECTIVE: Trauma is the most common cause of death among young adults. Alcohol intoxication plays a significant role as a cause of accidents and as a potent immunomodulator of the post-traumatic response to tissue injury. Polytraumatized patients are frequently at risk to developing infectious complications, which may be aggravated by alcohol-induced immunosuppression. Systemic levels of integral proteins of the gastrointestinal tract such as syndecan-1 or intestinal fatty acid binding proteins (FABP-I) reflect the intestinal barrier function. The exact impact of acute alcohol intoxication on the barrier function and endotoxin bioactivity have not been clarified yet. METHODS: 22 healthy volunteers received a precisely defined amount of alcohol (whiskey-cola) every 20 min over a period of 4 h to reach the calculated blood alcohol concentration (BAC) of 1‰. Blood samples were taken before alcohol drinking as a control, and after 2, 4, 6, 24 and 48 h after beginning with alcohol consumption. In addition, urine samples were collected. Intestinal permeability was determined by serum and urine values of FABP-I, syndecan-1, and soluble (s)CD14 as a marker for the endotoxin translocation via the intestinal barrier by ELISA. BAC was determined. RESULTS: Systemic FABP-I was significantly reduced 2 h after the onset of alcohol drinking, and remained decreased after 4 h. However, at 6 h, FABP-I significantly elevated compared to previous measurements as well as to controls (p < 0.05). Systemic sCD14 was significantly elevated after 6, 24 and 48 h after the onset of alcohol consumption (p < 0.05). Systemic FABP-I at 2 h after drinking significantly correlated with the sCD14 concentration after 24 h indicating an enhanced systemic LPS bioactivity. Women showed significantly lower levels of syndecan-1 in serum and urine and urine for all time points until 6 h and lower FABP-I in the serum after 2 h. CONCLUSIONS: Even relative low amounts of alcohol affect the immune system of healthy volunteers, although these changes appear minor in women. A potential damage to the intestinal barrier and presumed enhanced systemic endotoxin bioactivity after acute alcohol consumption is proposed, which represents a continuous immunological challenge for the organism and should be considered for the following days after drinking.

Enhanced Susceptibility of ADAP-Deficient Mice to Listeria monocytogenes Infection Is Associated With an Altered Phagocyte Phenotype and Function.

The adhesion and degranulation-promoting adaptor protein (ADAP) serves as a multifunctional scaffold and is involved in the formation of immune signaling complexes. To date, only limited data exist regarding the role of ADAP in pathogen-specific immunity during in vivo infection, and its contribution in phagocyte-mediated antibacterial immunity remains elusive. Here, we show that mice lacking ADAP (ADAPko) are highly susceptible to the infection with the intracellular pathogen Listeria monocytogenes (Lm) by showing enhanced immunopathology in infected tissues together with increased morbidity, mortality, and excessive infiltration of neutrophils and monocytes. Despite high phagocyte numbers in the spleen and liver, ADAPko mice only inefficiently controlled pathogen growth, hinting at a functional impairment of infection-primed phagocytes in the ADAP-deficient host. Flow cytometric analysis of hallmark pro-inflammatory mediators and unbiased whole genome transcriptional profiling of neutrophils and inflammatory monocytes uncovered broad molecular alterations in the inflammatory program in both phagocyte subsets following their activation in the ADAP-deficient host. Strikingly, ex vivo phagocytosis assay revealed impaired phagocytic capacity of neutrophils derived from Lm-infected ADAPko mice. Together, our data suggest that an alternative priming of phagocytes in ADAP-deficient mice during Lm infection induces marked alterations in the inflammatory profile of neutrophils and inflammatory monocytes that contribute to enhanced immunopathology while limiting their capacity to eliminate the pathogen and to prevent the fatal outcome of the infection.

Influenza A Virus (H1N1) Infection Induces Microglial Activation and Temporal Dysbalance in Glutamatergic Synaptic Transmission.

Influenza A virus (IAV) causes respiratory tract disease and is responsible for seasonal and reoccurring epidemics affecting all age groups. Next to typical disease symptoms, such as fever and fatigue, IAV infection has been associated with behavioral alterations presumably contributing to the development of major depression. Previous experiments using IAV/H1N1 infection models have shown impaired hippocampal neuronal morphology and cognitive abilities, but the underlying pathways have not been fully described. In this study, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes ample peripheral immune response followed by a temporary blood-brain barrier disturbance. Although histological examination did not reveal obvious pathological processes in the brains of IAV-infected mice, detailed multidimensional flow cytometric characterization of immune cells uncovered subtle alterations in the activation status of microglial cells. More specifically, we detected an altered expression pattern of major histocompatibility complex classes I and II, CD80, and F4/80 accompanied by elevated mRNA levels of CD36, CD68, C1QA, and C3, suggesting evolved synaptic pruning. To closer evaluate how these profound changes affect synaptic balance, we established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry. The introduction of this novel technique enabled us to simultaneously quantify the abundance of pre- and postsynapses from distinct brain regions. Our data reveal a significant reduction of VGLUT1 in excitatory presynaptic terminals in the cortex and hippocampus, identifying a subtle dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations. IMPORTANCE Influenza A virus (IAV) causes mainly respiratory tract disease with fever and fatigue but is also associated with behavioral alterations in humans. Here, we demonstrate that infection with a low-dose non-neurotrophic H1N1 strain of IAV causes peripheral immune response followed by a temporary blood-brain barrier disturbance. Characterization of immune cells uncovered subtle alterations in the activation status of microglia cells that might reshape neuronal synapses. We established a highly sensitive multiplex flow cytometry-based approach called flow synaptometry to more closely study the synapses. Thus, we detected a specific dysbalance in glutamatergic synapse transmission upon H1N1 infection in mice. In conclusion, our results highlight the consequences of systemic IAV-triggered inflammation on the central nervous system and the induction and progression of neuronal alterations.

Acute Alcohol Intoxication Modulates Monocyte Subsets and Their Functions in a Time-Dependent Manner in Healthy Volunteers.

Background: Excessive alcohol intake is associated with adverse immune response-related effects, however, acute and chronic abuse differently modulate monocyte activation. In this study, we have evaluated the phenotypic and functional changes of monocytes in acutely intoxicated healthy volunteers (HV). Methods: Twenty-two HV consumed individually adjusted amounts of alcoholic beverages until reaching a blood alcohol level of 1‰ after 4h (T4). Peripheral blood was withdrawn before and 2h (T2), 4h (T4), 6h (T6), 24h (T24), and 48h (T48) after starting the experiment and stained for CD14, CD16 and TLR4. CD14brightCD16-, CD14brightCD16+ and CD14dimCD16+ monocyte subsets and their TLR4 expression were analyzed by flow cytometry. Inflammasome activation via caspase-1 in CD14+ monocytes was measured upon an ex vivo in vitro LPS stimulation. Systemic IL-1ß and adhesion capacity of isolated CD14+ monocytes upon LPS stimulation were evaluated. Results: The percentage of CD14+ monocyte did not change following alcohol intoxication, whereas CD14brightCD16- monocyte subset significantly increased at T2 and T24, CD14brightCD16+ at T2, T4 and T6 and CD14dimCD16+ at T4 and T6. The relative fraction of TLR4 expressing CD14+ monocytes as well as the density of TLR4 surface presentation increased at T2 and decreased at T48 significantly. TLR4+CD14+ monocytes were significantly enhanced in all subsets at T2. TLR4 expression significantly decreased in CD14brightCD16- at T48, in CD14brightCD16+ at T24 and T48, increased in CD14dimCD16+ at T2. IL-1ß release upon LPS stimulation decreased at T48, correlating with TLR4 receptor expression. Alcohol downregulated inflammasome activation following ex vivo in vitro stimulation with LPS between T2 and T48 vs. T0. The adhesion capacity of CD14+ monocytes decreased from T2 with significance at T4, T6 and T48. Following LPS administration, a significant reduction of adhesion was observed at T4 and T6. Conclusions: Alcohol intoxication immediately redistributes monocyte subsets toward the pro-inflammatory phenotype with their subsequent differentiation into the anti-inflammatory phenotype. This is paralleled by a significant functional depression, suggesting an alcohol-induced time-dependent hyporesponsiveness of monocytes to pathogenic triggers.

The Immunoproteasome Subunits LMP2, LMP7 and MECL-1 Are Crucial Along the Induction of Cerebral Toxoplasmosis.

Cell survival and function critically relies on the fine-tuned balance of protein synthesis and degradation. In the steady state, the standard proteasome is sufficient to maintain this proteostasis. However, upon inflammation, the sharp increase in protein production requires additional mechanisms to limit protein-associated cellular stress. Under inflammatory conditions and the release of interferons, the immunoproteasome (IP) is induced to support protein processing and recycling. In antigen-presenting cells constitutively expressing IPs, inflammation-related mechanisms contribute to the formation of MHC class I/II-peptide complexes, which are required for the induction of T cell responses. The control of Toxoplasma gondii infection relies on Interferon-γ (IFNγ)-related T cell responses. Whether and how the IP affects the course of anti-parasitic T cell responses along the infection as well as inflammation of the central nervous system is still unknown. To answer this question we used triple knockout (TKO) mice lacking the 3 catalytic subunits of the immunoproteasome (ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7). Here we show that the numbers of dendritic cells, monocytes and CD8+ T cells were reduced in Toxoplasma gondii-infected TKO mice. Furthermore, impaired IFNγ, TNF and iNOS production was accompanied by dysregulated chemokine expression and altered immune cell recruitment to the brain. T cell differentiation was altered, apoptosis rates of microglia and monocytes were elevated and STAT3 downstream signaling was diminished. Consequently, anti-parasitic immune responses were impaired in TKO mice leading to elevated T. gondii burden and prolonged neuroinflammation. In summary we provide evidence for a critical role of the IP subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 for the control of cerebral Toxoplasma gondii infection and subsequent neuroinflammation.

Alterations of Phagocytic Activity and Capacity in Granulocytes and Monocytes Depend on the Pathogen Strain in Porcine Polytrauma.

Background: Polytraumatized patients undergo a strong immunological stress upon insult. Phagocytes (granulocytes and monocytes) play a substantial role in immunological defense against bacteria, fungi and yeast, and in the clearance of cellular debris after tissue injury. We have reported a reduced monocytes phagocytic activity early after porcine polytrauma before. However, it is unknown if both phagocyte types undergo those functional alterations, and if there is a pathogen-specific phagocytic behavior. We characterized the phagocytic activity and capacity of granulocytes and monocytes after polytrauma. Methods: Eight pigs (Sus scrofa) underwent polytrauma consisting of lung contusion, liver laceration, tibial fracture and hemorrhagic shock with fluid resuscitation and fracture fixation with external fixator. Intensive care treatment including mechanical ventilation for 72 h followed. Phagocytic activity and capacity were investigated using an in vitro ex vivo whole blood stimulation phagocytosis assays before trauma, after surgery, 24, 48, and 72 h after trauma. Blood samples were stimulated with Phorbol-12-myristate-13-acetate and incubated with FITC-labeled E. coli, S. aureus or S. cerevisiae for phagocytosis assessment by flow cytometry. Results: Early polytrauma-induced significant increase of granulocytes and monocytes declined to baseline values within 24 h. Percentage of E. coli-phagocytizing granulocytes significantly decreased after polytrauma and during further intensive care treatment, while their capacity significantly increased. Interestingly, both granulocytic phagocytic activity and capacity of S. aureus significantly decreased after trauma, although a recovery was observed after 24 h and yet was followed by another decrease. The percentage of S. cerevisiae-phagocytizing granulocytes significantly increased after 24 h, while their impaired capacity after surgery and 72 h later was detected. Monocytic E. coli-phagocytizing percentage did not change, while their capacity increased after 24-72 h. After a significant decrease in S. aureus-phagocytizing monocytes after surgery, a significant increase after 24 and 48 h was observed without capacity alterations. No significant changes in S. cerevisiae-phagocytizing monocytes occurred, but their capacity dropped 48 and 72 h. Conclusion: Phagocytic activity and capacity of granulocytes and monocytes follow a different pattern and significantly change within 72 h after polytrauma. Both phagocytic activity and capacity show significantly different alterations depending on the pathogen strain, thus potentially indicating at certain and possibly more relevant infection causes after polytrauma.

Robustly High Hippocampal BDNF levels under Acute Stress in Mice Lacking the Full-length p75 Neurotrophin Receptor.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) exerts its effects on neural plasticity via 2 distinct receptor types, the tyrosine kinase TrkB and the p75 neurotrophin receptor (p75NTR). The latter can promote inflammation and cell death while TrkB is critically involved in plasticity and memory, particularly in the hippocampus. Acute and chronic stress have been associated with suppression of hippocampal BDNF expression and impaired hippocampal plasticity. We hypothesized that p75NTR might be involved in the hippocampal stress response, in particular in stress-induced BDNF suppression, which might be accompanied by increased neuroinflammation. METHOD: We assessed hippocampal BDNF protein concentrations in wild-type mice compared that in mice lacking the long form of the p75NTR (p75NTRExIII-/-) with or without prior exposure to a 1-hour restraint stress challenge. Hippocampal BDNF concentrations were measured using an optimized ELISA. Furthermore, whole-brain mRNA expression of pro-inflammatory interleukin-6 (Il6) was assessed with RT-PCR. RESULTS: Deletion of full-length p75NTR was associated with higher hippocampal BDNF protein concentration in the stress condition, suggesting persistently high hippocampal BDNF levels in p75NTR-deficient mice, even under stress. Stress elicited increased whole-brain Il6 mRNA expression irrespective of genotype; however, p75NTRExIII-/- mice showed elevated baseline Il6 expression and thus a lower relative increase. CONCLUSIONS: Our results provide evidence for a role of p75NTR signaling in the regulation of hippocampal BDNF levels, particularly under stress. Furthermore, p75NTR signaling modulates baseline but not stress-related Il6 gene expression in mice. Our findings implicate p75NTR signaling as a potential pathomechanism in BDNF-dependent modulation of risk for neuropsychiatric disorders.