Porcine Colostrum Protects the IPEC-J2 Cells and Piglet Colon Epithelium against Clostridioides (syn. Clostridium) difficile Toxin-Induced Effects

Clostridioides difficile toxins are one of the main causative agents for the clinical symptoms observed during C. difficile infection in piglets. Porcine milk has been shown to strengthen the epithelial barrier function in the piglet’s intestine and may have the potential to neutralise clostridial toxins. We hypothesised that porcine colostrum exerts protective effects against those toxins in the IPEC-J2 cells and in the colon epithelium of healthy piglets. The IPEC-J2 cells were treated with either the toxins or porcine colostrum or their combination. Analyses included measurement of trans-epithelial electrical resistance (TEER), cell viability using propidium iodide by flow cytometry, gene expression of tight junction (TJ) proteins and immune markers, immunofluorescence (IF) histology of the cytoskeleton and a TJ protein assessment. Colon tissue explants from one- and two-week-old suckling piglets and from five-week-old weaned piglets were treated with C. difficile toxins in Ussing chamber assays to assess the permeability to macromolecules (FITC-dextran, HRP), followed by analysis of gene expression of TJ proteins and immune markers. Toxins decreased viability and integrity of IPEC-J2 cells in a time-dependent manner. Porcine colostrum exerted a protective effect against toxins as indicated by TEER and IF in IPEC-J2 cells. Toxins tended to increase paracellular permeability to macromolecules in colon tissues of two-week-old piglets and downregulated gene expression of occludin in colon tissues of five-week-old piglets (p = 0.05). Porcine milk including colostrum, besides other maternal factors, may be one of the important determinants of early immune programming towards protection from C. difficile infections in the offspring

In the Right Place, at the Right Time: Spatiotemporal Conditions Determining Plasma Cell Survival and Function

Plasma cells (PCs), the B lineage cells responsible for producing and secreting antibodies (Abs), are critical cellular components of the humoral immune system. While most of the antibody-secreting cells in the body have a rather short lifetime of a few days, some of them can become long-lived and persist in the body over the entire life span of an individual. The majority of these long-lived plasma cells secretes protective antibodies against pathogens, and are thereby crucial for the humoral component of immunological memory. The generation of these protective antibody-secreting cells can be triggered by an exposure to pathogens, and also by vaccination. Although the majority of plasma cells are protective, sometimes long-lived plasma cells produce autoreactive antibodies, which contribute to the pathogenesis and perpetuation of chronic autoimmune diseases, including lupus erythematosus, rheumatoid arthritis, or multiple sclerosis. In order to promote the formation of protective antibody-secreting cells and to target pathogenic plasma cells, it is crucial to understand the signals which promote their longevity and allow them to exert their function. In recent years, it has become clear that plasma cells depend on extrinsic factors for their survival, leading to the concept that certain tissue microenvironments promote plasma cell retention and longevity. However, these niches are not static structures, but also have dynamic features with respect to their cellular composition. Here, we review what is known about the molecular and cellular composition of the niches, and discuss the impact of dynamic changes within these microenvironments on plasma cell function. As plasma cell metabolism is tightly linked to their function, we present new tools, which will allow us to analyze metabolic parameters in the plasma cell niches in vivo over time

Analyzing nicotinamide adenine dinucleotide phosphate oxidase activation in aging and vascular amyloid pathology

In aging individuals, both protective as well as regulatory immune functions are declining, resulting in an increased susceptibility to infections as well as to autoimmunity. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2-deficiency in immune cell subsets has been shown to be associated with aging. Using intravital marker-free NAD(P)H-fluorescence lifetime imaging, we have previously identified microglia/myeloid cells and astrocytes as main cellular sources of NADPH oxidase (NOX) activity in the CNS during neuroinflammation, due to an overactivation of NOX. The overactivated NOX enzymes catalyze the massive production of the highly reactive O−2, which initiates in a chain reaction the overproduction of diverse reactive oxygen species (ROS). Age-dependent oxidative distress levels in the brain and their cellular sources are not known. Furthermore, it is unclear whether in age- dependent diseases oxidative distress is initiated by overproduction of ROS or by a decrease in antioxidant capacity, subsequently leading to neurodegeneration in the CNS. Here, we compare the activation level of NOX enzymes in the cerebral cortex of young and aged mice as well as in a model of vascular amyloid pathology. Despite the fact that a striking change in the morphology of microglia can be detected between young and aged individuals, we find comparable low-level NOX activation both in young and old mice. In contrast, aged mice with the human APPE693Q mutation, a model for cerebral amyloid angiopathy (CAA), displayed increased focal NOX overactivation in the brain cortex, especially in tissue areas around the vessels. Despite activated morphology in microglia, NOX overactivation was detected only in a small fraction of these cells, in contrast to other pathologies with overt inflammation as experimental autoimmune encephalomyelitis (EAE) or glioblastoma. Similar to these pathologies, the astrocytes majorly contribute to the NOX overactivation in the brain cortex during CAA. Together, these findings emphasize the role of other cellular sources of activated NOX than phagocytes not only during EAE but also in models of amyloid pathology. Moreover, they may strengthen the hypothesis that microglia/monocytes show a diminished potential for clearance of amyloid beta protein

Ongoing Oxidative Stress Causes Subclinical Neuronal Dysfunction in the Recovery Phase of EAE

Most multiple sclerosis (MS) patients develop over time a secondary progressive disease course, characterized histologically by axonal loss and atrophy. In early phases of the disease, focal inflammatory demyelination leads to functional impairment, but the mechanism of chronic progression in MS is still under debate. Reactive oxygen species generated by invading and resident central nervous system (CNS) macrophages have been implicated in mediating demyelination and axonal damage, but demyelination and neurodegeneration proceed even in the absence of obvious immune cell infiltration, during clinical recovery in chronic MS. Here, we employ intravital NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX1–4, DUOX1, 2) and, thus, to identify the cellular source of oxidative stress in the CNS of mice affected by experimental autoimmune encephalomyelitis (EAE) in the remission phase of the disease. This directly affects neuronal function in vivo, as monitored by cellular calcium levels using intravital FRET–FLIM, providing a possible mechanism of disease progression in MS

Low-Density Granulocytes Are a Novel Immunopathological Feature in Both Multiple Sclerosis and Neuromyelitis Optica Spectrum Disorder

Objective: To investigate whether low-density granulocytes (LDGs) are an immunophenotypic feature of patients with multiple sclerosis (MS) or neuromyelitis optica spectrum disorder (NMOSD). Methods: Blood samples were collected from 20 patients with NMOSD and 17 patients with MS, as well as from 15 patients with Systemic Lupus Erythematosus (SLE) and 23 Healthy Donors (HD). We isolated peripheral blood mononuclear cells (PBMCs) with density gradient separation and stained the cells with antibodies against CD14, CD15, CD16, and CD45, and analyzed the cells by flow cytometry or imaging flow cytometry. We defined LDGs as CD14-CD15(high) and calculated their share in total PBMC leukocytes (CD45+) as well as the share of CD16(hi) LDGs. Clinical data on disease course, medication, and antibody status were obtained. Results: LDGs were significantly more common in MS and NMOSD than in HDs, comparable to SLE samples (median values HD 0.2%, MS 0.9%, NMOSD 2.1%, SLE 4.3%). 0/23 of the HDs, but 17/20 NMOSD and 11/17 MS samples as well as 13/15 SLE samples had at least 0.7 % LDGs. NMOSD patients without continuous immunosuppressive treatment had significantly more LDGs compared to their treated counterparts. LDG nuclear morphology ranged from segmented to rounded, suggesting a heterogeneity within the group. Conclusion: LDGs are a feature of the immunophenotype in some patients with MS and NMOSD

Spatial Distribution of Macrophages During Callus Formation and Maturation Reveals Close Crosstalk Between Macrophages and Newly Forming Vessels

Macrophages are essential players in the process of fracture healing, acting by remodeling of the extracellular matrix and enabling vascularization. Whilst activated macrophages of M1-like phenotype are present in the initial pro-inflammatory phase of hours to days of fracture healing, an anti-inflammatory M2-like macrophage phenotype is supposed to be crucial for the induction of downstream cascades of healing, especially the initiation of vascularization. In a mouse-osteotomy model, we provide a comprehensive characterization of vessel (CD31+, Emcn+) and macrophage phenotypes (F4/80, CD206, CD80, Mac-2) during the process of fracture healing. To this end, we phenotype the phases of vascular regeneration-the expansion phase (d1-d7 after injury) and the remodeling phase of the endothelial network, until tissue integrity is restored (d14-d21 after injury). Vessels which appear during the bone formation process resemble type H endothelium (CD31hiEmcnhi), and are closely connected to osteoprogenitors (Runx2+, Osx+) and F4/80+ macrophages. M1-like macrophages are present in the initial phase of vascularization until day 3 post osteotomy, but they are rare during later regeneration phases. M2-like macrophages localize mainly extramedullary, and CD206+ macrophages are found to express Mac-2+ during the expansion phase. VEGFA expression is initiated by CD80+ cells, including F4/80+ macrophages, until day 3, while subsequently osteoblasts and chondrocytes are main contributors to VEGFA production at the fracture site. Using Longitudinal Intravital Microendoscopy of the Bone (LIMB) we observe changes in the motility and organization of CX3CR1+ cells, which infiltrate the injury site after an osteotomy. A transient accumulation, resulting in spatial polarization of both, endothelial cells and macrophages, in regions distal to the fracture site, is evident. Immunofluorescence histology followed by histocytometric analysis reveals that F4/80+CX3CR1+ myeloid cells precede vascularization

Teriflunomide Preserves Neuronal Activity and Protects Mitochondria in Brain Slices Exposed to Oxidative Stress

Teriflunomide (TFN) limits relapses in relapsing-remitting multiple sclerosis (RRMS) by reducing lymphocytic proliferation through the inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH) and the subsequent modulation of de novo pyrimidine synthesis. Alterations of mitochondrial function as a consequence of oxidative stress have been reported during neuroinflammation. Previously, we showed that TFN prevents alterations of mitochondrial motility caused by oxidative stress in peripheral axons. Here, we aimed to validate TFN effects on mitochondria and neuronal activity in hippocampal brain slices, in which cellular distribution and synaptic circuits are largely preserved. TFN effects on metabolism and neuronal activity were investigated by assessing oxygen partial pressure and local field potential in acute slices. Additionally, we imaged mitochondria in brain slices from the transgenic Thy1-CFP/COX8A)S2Lich/J (mitoCFP) mice using two-photon microscopy. Although TFN could not prevent oxidative stress-related depletion of ATP, it preserved oxygen consumption and neuronal activity in CNS tissue during oxidative stress. Furthermore, TFN prevented mitochondrial shortening and fragmentation of puncta-shaped and network mitochondria during oxidative stress. Regarding motility, TFN accentuated the decrease in mitochondrial displacement and increase in speed observed during oxidative stress. Importantly, these effects were not associated with neuronal viability and did not lead to axonal damage. In conclusion, during conditions of oxidative stress, TFN preserves the functionality of neurons and prevents morphological and motility alterations of mitochondria

Teriflunomide preserves peripheral nerve mitochondria from oxidative stress-mediated alterations

Mitochondrial dysfunction is a common pathological hallmark in various inflammatory and degenerative diseases of the central nervous system, including multiple sclerosis (MS). We previously showed that oxidative stress alters axonal mitochondria, limiting their transport and inducing conformational changes that lead to axonal damage. Teriflunomide (TFN), an oral immunomodulatory drug approved for the treatment of relapsing forms of MS, reversibly inhibits dihydroorotate dehydrogenase (DHODH). DHODH is crucial for de novo pyrimidine biosynthesis and is the only mitochondrial enzyme in this pathway, thus conferring a link between inflammation, mitochondrial activity and axonal integrity. Here, we investigated how DHODH inhibition may affect mitochondrial behavior in the context of oxidative stress. We employed a model of transected murine spinal roots, previously developed in our laboratory. Using confocal live imaging of axonal mitochondria, we showed that in unmanipulated axons, TFN increased significantly the mitochondria length without altering their transport features. In mitochondria challenged with 50 µM hydrogen peroxide (H2O2) to induce oxidative stress, the presence of TFN at 1 µM concentration was able to restore mitochondrial shape, motility, as well as mitochondrial oxidation potential to control levels. No effects were observed at 5 µM TFN, while some shape and motility parameters were restored to control levels at 50 µM TFN. Thus, our data demonstrate an undescribed link between DHODH and mitochondrial dynamics and point to a potential neuroprotective effect of DHODH inhibition in the context of oxidative stress-induced damage of axonal mitochondria