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

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.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.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-1β. 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.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

The Central Nervous System Contains ILC1s That Differ From NK Cells in the Response to Inflammation

Innate lymphoid cells (ILCs) are tissue resident cells with organ-specific properties. Here, we show that the central nervous system (CNS) encompasses ILCs. In particular, CD3-NK1.1+ cells present in the murine CNS comprise natural killer (NK) cells, ILC1s, intermediate ILC1s (intILC1s) and ex-ILC3s. We investigated the properties of CNS-ILC1s in comparison with CNS-NK cells during steady state and experimental autoimmune encephalomyelitis (EAE). ILC1s characteristically express CXCR3, CXCR6, DNAM-1, TRAIL, and CD200R and display heightened TNF-α production upon stimulation. In addition, ILC1s express perforin and are able to degranulate, although in a lesser extent than NK cells. Within the CNS compartments, ILC1s are enriched in the choroid plexus where very few NK cells are present, and also reside in the brain parenchyma and meninges. During EAE, ILC1s maintain stable IFN-γ and TNF-α levels while in NK cells the production of these cytokines increases as EAE progresses. Moreover, the amount of ILC1s and intILC1s increase in the parenchyma during EAE, but in contrast to NK cells, they show no signs of local proliferation. The upregulation in the inflamed brain of chemokines involved in ILC1 migration, such as CXCL9, CXCL10, and CXCL16 may lead to a recruitment of ILC1s from meninges or choroid plexus into the brain parenchyma. In sum, CNS-ILC1 phenotype, distribution and moderate inflammatory response during EAE suggest that they may act as gatekeepers involved in the control of neuroinflammation

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

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

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

Immune response to Toxoplasma gondii at the choroid plexus and the the immunomodulatory impact of the neuropeptide PACAP

Toxoplasma gondii (T. gondii) is a highly successful parasite able to cross all biological barriers of the body. The parasites invade through the intestinal epithelium the body, where they cause acute infection, thereafter they cross the borders of the central nervous system (CNS), entering the brain and inducing neuroinflammation. Previous studies have described the critical involvement of the CNS borders during T. gondii invasion and the development of neuroinflammation. While many studies have focused on the blood-brain barrier (BBB), the contribution of the choroid plexus (CP), the main structure forming the blood-cerebrospinal fluid (CSF)-barrier (BCSFB) has remained largely unknown. In this present study, confocal imaging of immuno-stained brain sections, tissue whole mount preparations, and determination of T. gondii gDNA levels were applied to reveal that CP is infected by T. gondii. In addition, flow cytometric and gene expression analysis showed that CP elicits an immune response prior to the BBB. Moreover, imaging and gene expression of tight junctions (TJ) in CP and primary cultured CP epithelial cells indicate a loss of function and integrity of the BCSFB, which was confirmed by in vivo FITC-dextran permeability assay [Publication 1]. Generally, albeit the elicited immune response during infection is essential for parasite control, continuous activation and recruitment of immune cells to infected organs entail tissue damage and cause detrimental alterations to their function. To mitigate these alterations, this study additionally set out to investigate the effects of the neuropeptide PACAP upon acute [Publication 2] and chronic T. gondii infection [Publication 3], as PACAP has shown immunomodulatory and neuroprotective properties in other disease models. Here, the infection of mice with GFP-reporter parasites indicate that PACAP displayed anti-parasitic activity, and further in vitro assays showed that this is an immune cell-mediated effect rather than a direct elimination of the parasites. Overall, flow cytometric and gene expression analysis showed that PACAP treatment reduced immune cell infiltration, activation, and cytokine production in the periphery and in the CNS. Additionally, a cross-talk between PACAP and neurotrophin signaling pathways was suggested to affect the immune as well as the nervous systems, and to promote neuroprotection during T. gondii infection. Altogether, those findings 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. Moreover, this study highlights the beneficial effects of PACAP application upon acute and chronic toxoplasmosis, providing new insights into the potential use of neuropeptides to counteract infection-induced inflammation