Immunomodulatory Effects of the Neuropeptide Pituitary Adenylate Cyclase-Activating Polypeptide in Acute Toxoplasmosis

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is an endogenous neuropeptide with distinct functions including the regulation of inflammatory processes. PACAP is able to modify the immune response by directly regulating macrophages and monocytes inhibiting the production of inflammatory cytokines, chemokines and free radicals. Here, we analyzed the effect of exogenous PACAP on peripheral immune cell subsets upon acute infection with the parasite Toxoplasma gondii (T. gondii). PACAP administration was followed by diminished innate immune cell recruitment to the peritoneal cavity of T. gondii-infected mice. PACAP did not directly interfere with parasite replication, instead, indirectly reduced parasite burden in mononuclear cell populations by enhancing their phagocytic capacity. Although proinflammatory cytokine levels were attenuated in the periphery upon PACAP treatment, interleukin (IL)-10 and Transforming growth factor beta (TGF-β) remained stable. While PACAP modulated VPAC1 and VPAC2 receptors in immune cells upon binding, it also increased their expression of brain-derived neurotrophic factor (BDNF). In addition, the expression of p75 neurotrophin receptor (p75NTR) on Ly6Chi inflammatory monocytes was diminished upon PACAP administration. Our findings highlight the immunomodulatory effect of PACAP on peripheral immune cell subsets during acute Toxoplasmosis, providing new insights about host-pathogen interaction and the effects of neuropeptides during inflammation

A murine intestinal intraepithelial NKp46-negative innate lymphoid cell population characterized by group 1 properties

The Ly49E receptor is preferentially expressed on murine innate-like lymphocytes, such as epidermal V gamma 3 T cells, intestinal intraepithelial CD8 alpha alpha(+) T lymphocytes, and CD49a(+) liver natural killer (NK) cells. As the latter have recently been shown to be distinct from conventional NK cells and have innate lymphoid cell type 1 (ILC1) properties, we investigated Ly49E expression on intestinal ILC populations. Here, we show that Ly49E expression is very low on known ILC populations, but it can be used to define a previously unrecognized intraepithelial innate lymphoid population. This Ly49E-positive population is negative for NKp46 and CD8 alpha alpha, expresses CD49a and CD103, and requires T-bet expression and IL-15 signaling for differentiation and/or survival. Transcriptome analysis reveals a group 1 ILC gene profile, different from NK cells, iCD8 alpha cells, and intraepithelial ILC1. Importantly, NKp46(-)CD8 alpha alpha(-)Ly49E(+) cells produce interferon (IFN)-gamma, suggesting that this previously unrecognized population may contribute to Th1-mediated immunity

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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12974-021-02370-1

Interferon-γ Receptor Signaling in Dendritic Cells Restrains Spontaneous Proliferation of CD4+ T Cells in Chronic Lymphopenic Mice

In lymphopenic mice, T cells become activated and undergo lymphopenia-induced proliferation (LIP). However, not all T cells are equally sensitive to lymphopenia. Several lymphopenia-insensitive T cell clones were described and their non-responsiveness was mainly attributed to clone-specific properties. Here, we provide evidence for an additional, host-dependent mechanism restraining LIP of lymphopenia-insensitive CD4+ T cells. We show that such cells undergo LIP in lymphopenic mice lacking IFN-γ receptor (IFN-γR) expression, a process, which is promoted by the autocrine action of T cell-derived IFN-γ. Additionally, LIP of lymphopenia-insensitive CD4+ T cells requires an intact microflora and is accompanied by the massive accumulation of IL-6 and dendritic cells (DCs). Consistent with these results, IL-6 neutralization and the DC-specific restoration of IFN-γR expression are both sufficient to restrict LIP. Hence, the insensitivity of CD4+ T cells to lymphopenia relies on cell-intrinsic properties and a complex interplay between the commensal microflora, IL-6, IFN-γR+ DCs, and T cell-derived IFN-γ

p75NTR regulates brain mononuclear cell function and neuronal structure in Toxoplasma infection-induced neuroinflammation.

Neurotrophins mediate neuronal growth, differentiation, and survival via tropomyosin receptor kinase (Trk) or p75 neurotrophin receptor (p75NTR ) signaling. The p75NTR is not exclusively expressed by neurons but also by certain immune cells, implying a role for neurotrophin signaling in the immune system. In this study, we investigated the effect of p75NTR on innate immune cell behavior and on neuronal morphology upon chronic Toxoplasma gondii (T. gondii) infection-induced neuroinflammation. Characterization of the immune cells in the periphery and central nervous system (CNS) revealed that innate immune cell subsets in the brain upregulated p75NTR upon infection in wild-type mice. Although cell recruitment and phagocytic capacity of p75NTRexonIV knockout (p75-/- ) mice were not impaired, the activation status of resident microglia and recruited myeloid cell subsets was altered. Importantly, recruited mononuclear cells in brains of infected p75-/- mice upregulated the production of the cytokines interleukin (IL)-10, IL-6 as well as IL-1α. Protein levels of proBDNF, known to negatively influence neuronal morphology by binding p75NTR , were highly increased upon chronic infection in the brain of wild-type and p75-/- mice. Moreover, upon infection the activated immune cells contributed to the proBDNF release. Notably, the neuroinflammation-induced changes in spine density were rescued in the p75-/- mice. In conclusion, these findings indicate that neurotrophin signaling via the p75NTR affects innate immune cell behavior, thus, influencing the structural plasticity of neurons under inflammatory conditions

Treatment of toxoplasmic encephalitis, in-vitro and in-vivo models to study passage of antiparasitic drugs through the blood-brain barrier

Titelblatt, Danksagung und Inhalt 1\. Einleitung 2\. Problemstellung und Zielsetzung der Arbeit 3\. Material und Methoden 4\. Ergebnisse und Diskussion. 4.1. In-vitro-Untersuchungen mit Zellen der BHS 4.2. In-vivo-Untersuchungen im Infektionsmodell der Reaktivierungstoxoplasmose 5\. Zusammenfassung 6\. Summary 7\. Abkürzungsverzeichnis 8\. Literaturverzeichnis 9\. Curriculum vitae 10\. Publikationen und KongressbeiträgeT. gondii verursacht eine in der Regel asymptomatisch verlaufende Infektion, die durch Persistenz des Erregers in Zysten vor allem im zentralen Nervensystem charakterisiert ist. Unter Immunsuppression (z.B. AIDS-Patienten, Transplantierte) kommt es zu einer Reaktivierung der Infektion, die unbehandelt unter dem Bild einer Enzephalitis tödlich verläuft. Die Therapie der Reaktivierungstoxoplasmose, einer der häufigsten zerebralen Infektionen bei Immunsupprimierten, setzt sich aus einer mindestens vierwöchigen Akut- Behandlung, gefolgt von einer lebenslang durchzuführenden Erhaltungstherapie, zusammen. Standardtherapeutika wie Pyrimethamin und Sulfadiazin sind durch hohe Toxizität oder Allergisierung gekennzeichnet; alternative Wirkstoffe werden nur wenig resorbiert und/oder sind wenig gehirngängig. Die Wirkstoffapplikation in Form von oberflächenmodifizierten Nanosuspensionen ermöglicht das gezielte Targeting von Zellen im zentralen Nervensystem. Um die Passage von Wirkstoff-Nanosuspensionen durch die Blut-Hirn-Schranke zu untersuchen, wurde in der vorliegenden Arbeit ein In-vitro-Modell der Blut- Hirn-Schranke etabliert. Die Kokultur von primären Ratten-Hirnendothelzellen mit primären Rattenastrozyten in einem Transwell-System war der Monokultur von unterschiedlichen Zellinien aus Maus oder Ratte überlegen. Sowohl die Messung des TEER (bis zu 350 Ohm x cm2) als auch die Permeabilitätsuntersuchungen bestätigten die grundsätzliche Eignung für Transportstudien. Problematisch stellte sich die Testung von mit Tween-80-beladenen Nanosuspensionen heraus, da diese selbst im zellfreien System vermutlich aufgrund von Aggregation oder Adhäsion an der Plastikoberfläche den Filter nicht dosisabhängig passierten. Um die Interaktion von T. gondii mit der Blut-Hirn-Schranke zu untersuchen, wurde die Infektion von primären Hirnendothelzellen der Ratte mit T. gondii mittels Laser-Scanning-Mikroskopie untersucht. Parallel dazu wurden zytotoxische Effekte des Parasiten auf Hirnendothelzellen mit verschiedenen Methoden untersucht. Es zeigte sich, dass Hirnendothelzellen innerhalb weniger Minuten von T. gondii infiziert wurden; deutliche zytotoxische Effekte stellten sich erst bei Parasiten-Zell-Verhältnissen von >1:1 ein. Um die Wirksamkeit von Antiparasitika-Nanosuspensionen zu untersuchen, wurden Tween-80-beladene Nanosuspensionen des Hydronaphthochinons Atovaquon (mit exzellenter In-vitro-Wirksamkeit gegen T. gondii) für die i.v.-Therapie hergestellt. Die Dosis und Häufigkeit der Verabreichung in der Akutphase der Behandlung wurde optimiert. Dabei wurden Konzentrationen im Serum der Versuchstiere von Atovaquon erreicht, die denen im Serum von oral-behandelten Patienten entsprechen. Darüber hinaus wurde erstmals ein Mausmodell der Reaktivierungstoxoplasmose zur Testung von Wirkstoffen für die Erhaltungstherapie etabliert. In diesem Modell zeigte eine oral verabreichte Atovaquon-Suspension eine der Standard-Erhaltungstherapie (Kombination von Pyrimethamin und Sulfadiazin) überlegene Wirksamkeit. Sowohl die Überlebenszeiten der behandelten Mäuse als auch die histologischen Veränderungen im Hirn und anderen Organen belegten die ausgezeichnete Wirksamkeit der Atovaquon-Suspension. Atovaquon konnte in Serum, Leber und Lunge der behandelten Tiere mittels HPLC, im Gehirn mittels Massenspektrometrie nachgewiesen werden. Die Ergebnisse der vorliegenden Dissertationsarbeit erlauben weiterführende detaillierte Untersuchungen zur Passage von Wirkstoffen durch die Blut-Hirn-Schranke im Kokultur-Modell. Darüber hinaus kann die Transmigration des Parasiten durch die Blut-Hirn- Schranke und die Interaktion mit Zellen der Blut-Hirn-Schranke in-vitro analysiert werden. Das Mausmodell der Reaktivierungstoxoplasmose ermöglicht in enger Anlehnung an die Situation bei immunsupprimierten Patienten die Testung des therapeutischen Effekts weiterer antiparasitärer Substanzen in der Akut- und Erhaltungstherapie. Atovaquon sollte in klinischen Studien zur Akut- und Erhaltungstherapie der Toxoplasma-Enzephalitis getestet werden.Primary infection with Toxoplasma gondii usually does not cause clinical symptoms. Infection is characterized by persistence of the parasite in cysts in the central nervous system. In immunocompromized hosts (i.e. AIDS- and transplanted patients) reactivation may occur and results in development of toxoplasmic encephalitis. Reactivation takes a lethal course if left untreated. Therapy comprises a 4-week course of acute treatment and lifelong maintenance treatment. Standard therapy regimens often cause allergy or have toxic side effects. Alternative therapy regimens show low bioavailability and/or do only poorly penetrate the blood-brain-barrier (BBB). Antiparasitic drugs prepared as nanosuspenions with modified surfaces (i.e. tween 80-coated) allow targeting of the central nervous system. To investigate the passage of antiparasitic nanosuspensions through the BBB, we established a co-culture transwell model of the BBB using primary rat brain endothelial cells and primary rat astrocytes. This co-culture model was superior to the commonly applied models using mouse or rat brain endothelial cell lines. We observed increased electrical resistance (TEER of up to 350 Ohm x cm2) as well as low paracellular permeability using sodium fluorescein as a marker. However, the passage of tween 80-coated nanosuspensions through the transwell system, was not dose dependent, most likely caised by aggregation of nanosuspensions and adherence to the polycarbonate filters was observed. To study the interaction of T. gondii tachyzoites with the BBB, we followed the infection of primary brain endothelial cells by T. gondii using laser-scanning microscopy. We observed rapid entry of the parasites into brain endothelial cells. In parasite-cell-ratios of >1:1, parasites showed dose-dependent cytotoxic effects on endothelial cells. Tween 80-coated nanosuspensions of the lipophilic hydronaphthochinone atovaquone (showing excellent in-vitro activity against T. gondii) were prepared to investigate targeting of the brain in- vivo. Dosage, time, and frequency of i.v.-administration of atovaquone nanosuspensions for acute treatment were optimized in a murine model of reactivated toxoplasmosis. In addition, we expanded the murine model of acute therapy to study the therapeutic efficacy of drugs in maintenance therapy. Oral administration of atovaquone suspensions showed superior therapeutic efficacy compared to standard therapy using the combination of pyrimethamine and sulfadiazine. Time to death was prolonged and histological signs of toxoplasmic encephalitis were absent in mice orally treated with atovaquone. High concentrations of atovaquone were detectable in sera, liver, and lungs of mice using HPLC whereas in brains of mice atovaquone was only detectable by highly sensitive mass spectrometry. Results of the present study will allow further studies to follow passage of antiparasitic drugs through the BBB. The co-culture model also allows to track transmigration of the parasite through the BBB. The murine model of reactivated toxoplasmosis closely mimics the clinical situation in immunocompromized hosts and therefore allows testing of antiparasitic drugs for acute and maintenance therapy in-vivo. Atovaquone should be further tested in clinical trials for acute and maintenance treatment of toxoplasmic encephalitis

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

Regulatory T cells modulate CD4 proliferation after severe trauma via IL-10

Objective: Severely injured patients frequently develop an immunological imbalance following the traumatic insult, which might result in infectious complications evoked by a persisting immunosuppression. Regulatory T cells (Tregs) maintain the immune homeostasis by suppressing proinflammatory responses, however, their functionality after trauma is unclear. Here, we characterized the role of Tregs in regulating the proliferation of CD4+ lymphocytes in traumatized patients (TP). Methods: Peripheral blood was obtained daily from 29 severely injured TP (Injury Severity Score, ISS ≥16) for ten days following admission to the emergency department (ED). Ten healthy volunteers (HV) served as controls. The frequency and activity of Tregs were assessed by flow cytometry. Proliferation of CD4+ cells was analyzed either in presence or absence of Tregs, or after blocking of either IL-10 or IL-10R1. Results: The frequencies of CD4+CD25high and CD4+CD25+CD127− Tregs were significantly decreased immediately upon admission of TP to the ED and during the following 10 post-injury days. Compared with HV CD4+ T cell proliferation in TP increased significantly upon their admission and on the following days. As expected, CD4+CD25+CD127− Tregs reduced the proliferation of CD4+ cells in HV, nevertheless, CD4+ proliferation in TP was increased by Tregs. Neutralization of IL-10 as well as blocking the IL-10R1 increased further CD4+ T cell proliferation in Tregs-depleted cultures, thereby confirming an IL-10-mediated mechanism of IL-10-regulated CD4+ T cell proliferation. Neutralization of IL-10 in TP decreased CD4+ T cell proliferation in Tregs-depleted cultures, whereas blocking of the IL-10R1 receptor had no significant effects. Conclusions: The frequency of Tregs in the CD4+ T lymphocyte population is reduced after trauma; however, their inductiveness is increased. The mechanisms of deregulated influence of Tregs on CD4+ T cell proliferation are mediated via IL-10 but not via the IL-10R1

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