Proteomic analysis of plasma-derived extracellular vesicles: pre- and postprandial comparisons.

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.

Microglial sex differences in innate high anxiety and modulatory effects of minocycline.

Microglia modulate synaptic refinement in the central nervous system (CNS). We have previously shown that a mouse model with innate high anxiety-related behavior (HAB) displays higher CD68+ microglia density in the key regions of anxiety circuits compared to mice with normal anxiety-related behavior (NAB) in males, and that minocycline treatment attenuated the enhanced anxiety of HAB male. Given that a higher prevalence of anxiety is widely reported in females compared to males, little is known concerning sex differences at the cellular level. Herein, we address this by analyzing microglia heterogeneity and function in the HAB and NAB brains of both sexes. Single-cell RNA sequencing revealed ten distinct microglia clusters varied by their frequency and gene expression profile. We report striking sex differences, especially in the major microglia clusters of HABs, indicating a higher expression of genes associated with phagocytosis and synaptic engulfment in the female compared to the male. On a functional level, we show that female HAB microglia engulfed a greater amount of hippocampal vGLUT1+ excitatory synapses compared to the male. We moreover show that female HAB microglia engulfed more synaptosomes compared to the male HAB in vitro. Due to previously reported effects of minocycline on microglia, we finally administered oral minocycline to HABs of both sexes and showed a significant reduction in the engulfment of synapses by female HAB microglia. In parallel to our microglia-specific findings, we further showed an anxiolytic effect of minocycline on female HABs, which is complementary to our previous findings in the male HABs. Our study, therefore, identifies the altered function of synaptic engulfment by microglia as a potential avenue to target and resolve microglia heterogeneity in mice with innate high anxiety.

Spatio-temporal dynamics of microglia phenotype in human and murine cSVD: impact of acute and chronic hypertensive states.

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.

IFNAR signaling in fibroblastic reticular cells can modulate CD8+ memory fate decision.

CD8+ memory T cells (TM ) are crucial for long-term protection from infections and cancer. Multiple cell types and cytokines are involved in the regulation of CD8+ T cell responses and subsequent TM formation. Besides their direct antiviral effects, type I interferons (IFN-I) modulate CD8+ T cell immunity via their action on several immune cell subsets. However, it is largely unclear how nonimmune cells are involved in this multicellular network modulating CD8+ TM formation. Fibroblastic reticular cells (FRCs) form the 3D scaffold of secondary lymphoid organs, express the IFN-I receptor (IFNAR), and modulate adaptive immune responses. However, it is unclear whether and how early IFNAR signals in lymph node (LN) FRCs affect CD8+ TM differentiation. Using peptide vaccination and viral infection, we studied CD8+ TM differentiation in mice with an FRC-specific IFNAR deletion (FRCΔIFNAR ). We show here that the differentiation of CD8+ TCR-transgenic T cells into central memory cells (TCM ) is enhanced in peptide-vaccinated FRCΔIFNAR mice. Conversely, vesicular stomatitis virus infection of FRCΔIFNAR mice is associated with impaired TCM formation and the accumulation of vesicular stomatitis virus specific double-positive CD127hi KLRG-1hi effector memory T cells. In summary, we provide evidence for a context-dependent contribution of FRC-specific IFNAR signaling to CD8+ TM differentiation.

Lymphotoxin ß Receptor: a Crucial Role in Innate and Adaptive Immune Responses against Toxoplasma gondii.

The lymphotoxin ß receptor (LTßR) plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LTßR is crucial for surviving acute toxoplasmosis; however, until now, a functional analysis was largely incomplete. Here, we demonstrate that the LTßR is a key regulator required for the intricate balance of adaptive immune responses. Toxoplasma gondii-infected LTßR-deficient (LTßR-/-) mice show globally altered interferon-γ (IFN-γ) regulation, reduced IFN-γ-controlled host effector molecule expression, impaired T cell functionality, and an absent anti-parasite-specific IgG response, resulting in a severe loss of immune control of the parasites. Reconstitution of LTßR-/- mice with toxoplasma immune serum significantly prolongs survival following T. gondii infection. Notably, analysis of RNA-seq data clearly indicates a specific effect of T. gondii infection on the B cell response and isotype switching. This study uncovers the decisive role of the LTßR in cytokine regulation and adaptive immune responses to control T. gondii.

Toxoplasma gondii infection and its implications within the central nervous system.

Toxoplasma gondii is a parasite that infects a wide range of animals and causes zoonotic infections in humans. Although it normally only results in mild illness in healthy individuals, toxoplasmosis is a common opportunistic infection with high mortality in individuals who are immunocompromised, most commonly due to reactivation of infection in the central nervous system. In the acute phase of infection, interferon-dependent immune responses control rapid parasite expansion and mitigate acute disease symptoms. However, after dissemination the parasite differentiates into semi-dormant cysts that form within muscle cells and neurons, where they persist for life in the infected host. Control of infection in the central nervous system, a compartment of immune privilege, relies on modified immune responses that aim to balance infection control while limiting potential damage due to inflammation. In response to the activation of interferon-mediated pathways, the parasite deploys an array of effector proteins to escape immune clearance and ensure latent survival. Although these pathways are best studied in the laboratory mouse, emerging evidence points to unique mechanisms of control in human toxoplasmosis. In this Review, we explore some of these recent findings that extend our understanding for proliferation, establishment and control of toxoplasmosis in humans.

NK Cells Negatively Regulate CD8 T Cells to Promote Immune Exhaustion and Chronic Toxoplasma gondii Infection.

NK cells regulate CD4+ and CD8+ T cells in acute viral infection, vaccination, and the tumor microenvironment. NK cells also become exhausted in chronic activation settings. The mechanisms causing these ILC responses and their impact on adaptive immunity are unclear. CD8+ T cell exhaustion develops during chronic Toxoplasma gondii (T. gondii) infection resulting in parasite reactivation and death. How chronic T. gondii infection impacts the NK cell compartment is not known. We demonstrate that NK cells do not exhibit hallmarks of exhaustion. Their numbers are stable and they do not express high PD1 or LAG3. NK cell depletion with anti-NK1.1 is therapeutic and rescues chronic T. gondii infected mice from CD8+ T cell exhaustion dependent death, increases survival after lethal secondary challenge and alters cyst burdens in brain. Anti-NK1.1 treatment increased polyfunctional CD8+ T cell responses in spleen and brain and reduced CD8+ T cell apoptosis in spleen. Chronic T. gondii infection promotes the development of a modified NK cell compartment, which does not exhibit normal NK cell characteristics. NK cells are Ly49 and TRAIL negative and are enriched for expression of CD94/NKG2A and KLRG1. These NK cells are found in both spleen and brain. They do not produce IFNγ, are IL-10 negative, do not increase PDL1 expression, but do increase CD107a on their surface. Based on the NK cell receptor phenotype we observed NKp46 and CD94-NKG2A cognate ligands were measured. Activating NKp46 (NCR1-ligand) ligand increased and NKG2A ligand Qa-1b expression was reduced on CD8+ T cells. Blockade of NKp46 rescued the chronically infected mice from death and reduced the number of NKG2A+ cells. Immunization with a single dose non-persistent 100% protective T. gondii vaccination did not induce this cell population in the spleen, suggesting persistent infection is essential for their development. We hypothesize chronic T. gondii infection induces an NKp46 dependent modified NK cell population that reduces functional CD8+ T cells to promote persistent parasite infection in the brain. NK cell targeted therapies could enhance immunity in people with chronic infections, chronic inflammation and cancer.

c-FLIP is crucial for IL-7/IL-15-dependent NKp46+ ILC development and protection from intestinal inflammation in mice.

NKp46+ innate lymphoid cells (ILC) modulate tissue homeostasis and anti-microbial immune responses. ILC development and function are regulated by cytokines such as Interleukin (IL)-7 and IL-15. However, the ILC-intrinsic pathways translating cytokine signals into developmental programs are largely unknown. Here we show that the anti-apoptotic molecule cellular FLICE-like inhibitory protein (c-FLIP) is crucial for the generation of IL-7/IL-15-dependent NKp46+ ILC1, including conventional natural killer (cNK) cells, and ILC3. Cytokine-induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) precedes up-regulation of c-FLIP, which protects developing NKp46+ ILC from TNF-induced apoptosis. NKp46+ ILC-specific inactivation of c-FLIP leads to the loss of all IL-7/IL-15-dependent NKp46+ ILC, thereby inducing early-onset chronic colitis and subsequently microbial dysbiosis; meanwhile, the depletion of cNK, but not NKp46+ ILC1/3, aggravates experimental colitis. In summary, our data demonstrate a non-redundant function of c-FLIP for the generation of NKp46+ ILC, which protect T/B lymphocyte-sufficient mice from intestinal inflammation.

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.

Pituitary Adenylate Cyclase-Activating Polypeptide-A Neuropeptide as Novel Treatment Option for Subacute Ileitis in Mice Harboring a Human Gut Microbiota.

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) is well-known for its important functions in immunity and inflammation. Data regarding anti-inflammatory properties of PACAP in the intestinal tract are limited, however. In our present preclinical intervention study we addressed whether PACAP treatment could alleviate experimental subacute ileitis mimicking human gut microbiota conditions. Therefore, secondary abioitic mice were subjected to human fecal microbiota transplantation (FMT) and perorally infected with low-dose Toxoplasma gondii to induce subacute ileitis on day 0. From day 3 until day 8 post-infection, mice were either treated with synthetic PACAP38 or placebo. At day 9 post-infection, placebo, but not PACAP treated mice exhibited overt macroscopic sequelae of intestinal immunopathology. PACAP treatment further resulted in less distinct apoptotic responses in ileal and colonic epithelia that were accompanied by lower T cell numbers in the mucosa and lamina propria and less secretion of pro-inflammatory cytokines in intestinal ex vivo biopsies. Notably, ileitis-associated gut microbiota shifts were less distinct in PACAP as compared to placebo treated mice. Inflammation-ameliorating effects of PACAP were not restricted to the intestines, but could also be observed in extra-intestinal including systemic compartments as indicated by lower apoptotic cell counts and less pro-inflammatory cytokine secretion in liver and lungs taken from PACAP treated as compared to placebo control mice, which also held true for markedly lower serum TNF and IL-6 concentrations in the former as compared to the latter. Our preclinical intervention study provides strong evidence that synthetic PACAP alleviates subacute ileitis and extra-intestinal including systemic sequelae of T cell-driven immunopathology. These findings further support PACAP as a novel treatment option for intestinal inflammation including inflammatory bowel diseases (IBD).