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The meningeal space is an important structure in the brain borders, which provides immunosurveillance for the central nervous system, but the impact of infections on the meningeal immune landscape is far from being fully understood.Trypanosoma brucei, the causative agents of Human African Trypanosomiasis or sleeping sickness, accumulates in the meningeal spaces, inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, combining single cell transcriptomics and mass cytometry by time of flight (CyTOF), coupled within vivointerventions, we found that chronicT. bruceiinfection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges during chronic infection. These infection-induced ectopic structures are defined by the presence of ER-TR7+fibroblastic reticular cells (FRCs) and follicular dendritic cells (FDCs) that initiate a signalling cascade driving local T cell activation towards a T follicular helper (TFH)-like phenotype, as well as B cell class switching. Furthermore, the meningeal-resident plasma cells display activity typically associated with germinal centre reactions and are able to produce antibodies able to recognise mouse brain antigens, which correlates with cortical (and white matter) demyelination. Lastly, we found that systemic lymphotoxinb(LTb) signalling blockade led to a significant depletion of meningeal FDCs and autoreactive B cells, indicating that LTbsignalling is critical to induce and maintain local responses in the meninges. Taken together, we provide evidence that the meningeal immune response results in the acquisition of lymphoid tissue-like properties during chronicT. bruceiinfection. These findings have broader implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity, as observed in other demyelinating neurodegenerative disorders such as multiple sclerosis.

The meningeal space is an important structure in the brain borders, which provides immunosurveillance for the central nervous system, but the impact of infections on the meningeal immune landscape is far from being fully understood. The extracellular protozoan parasite Trypanosoma brucei, which causes Human African Trypanosomiasis (HAT) or sleeping sickness, accumulate in the meningeal spaces, ultimately inducing severe meningitis and resulting in death if left untreated. Thus, sleeping sickness represents an attractive model to study immunological dynamics in the meninges during infection. Here, combining single cell transcriptomics and mass cytometry by time of flight (CyTOF), coupled with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges during chronic infection. These infection-induced ectopic structures are defined by the presence of ER-TR7+ fibroblastic reticular cells (FRCs) and follicular dendritic cells (FDCs) that initiate a signalling cascade driving local T cell activation towards a T follicular helper (TFH)-like phenotype, as well as B cellclass switching. Furthermore, the GC-like B cells found in the infected meningesproduce high-affinity autoantibodies able to recognise mouse brain antigens. We found that systemic lymphotoxin β (LTβ) signalling blockade led to a significant depletion of meningeal FDC-like cells and autoreactive B cells, indicating that LTβ signalling is critical to induce and maintain local responses in the meninges. In humans, we identified the presence of autoreactive IgG antibodies able to recognise human brain lysates in the cerebrospinal fluid of second stage HAT patients compared to first stage HAT patients, consistent with our findings in experimental infections. Taken together, our data provide evidence that the meningeal immune response results in the acquisition of lymphoid tissue-like properties during chronic T. brucei infection, broadening our understanding of meningeal immunity in the context of chronic infections. These findings have wider implications for understanding the mechanisms underlying the formation ELAs during chronic inflammation resulting in autoimmunity in mice and humans, as observed in other autoimmune neurodegenerative disorders such as neuropsychiatric lupus and multiple sclerosis.

Microglia play key roles in brain homeostasis as well as responses to neurodegeneration and neuroinflammatory processes caused by physical disease and psychosocial stress. The pig is a physiologically-relevant model species for studying human neurological disorders, many of which are associated with microglial dysfunction. Furthermore, pigs are an important agricultural species, and there is a need to understand how microglial function affects their welfare. As a basis for improved understanding to enhance biomedical and agricultural research, we sought to characterise pig microglial identity at genome-wide scale and conduct inter-species comparisons. We isolated pig hippocampal tissue and microglia from frontal cortex, hippocampus and cerebellum, as well as alveolar macrophages from the lungs and conducted RNA-sequencing (RNAseq). By comparing the transcriptomic profiles between microglia, macrophages, and hippocampal tissue, we derived a set of 239 highly-enriched genes defining the porcine core microglial signature. We found brain regional heterogeneity based on 150 genes showing significant (adjusted pand demonstrated that a core microglial marker gene signature is conserved across species, but that species-specific expression subsets also exist.Our data provide a valuable resource defining the pig microglial transcriptome signature that validates and highlights pigs as a useful large animal species bridging between rodents and humans in which to study the role of microglia during homeostasis and disease.

Additional file 10. Leucocytes identified in the chicken enteroid lamina propria. Expression of immune cell population gene sets analysed by RNA sequencing analysis. Confocal images of chicken enteroids at 2 days of culture stained for CD45.

Additional file 2. Primers and probes used in RT-qPCR.

Additional file 7. Proliferation in the enteroids. Graph demonstrating the percentage of proliferating (EdU+) cells within enteroids over 7 days of culture, alongside z-stack image of proliferating (EdU+) cells within a 14 h enteroid.

Additional file 5. Mean expression profiles of the genes in each of the largest 50 co-expression clusters. Individual mean expression profiles of the genes in each of the largest 50 co-expression clusters derived from the network graph. The x axis shows the samples ordered by time of cultures. The y axis shows the mean expression intensity (transcripts/million reads, TPM) for the cluster.

Additional file 8. Cell-type gene expression in chicken enteroid cultures. Expression of stem cell, Paneth cell, enteroendocrine cell, goblet cell and enterocyte genes in 1 day chicken enteroid cultures. Confocal images of chicken enteroids at 2 days of culture stained for.

Additional file 3. Primary antibodies used for immunohistochemistry.

Prion diseases are transmissible, neurodegenerative disorders associated with misfolding of the prion protein. Previous studies show that reduction of microglia accelerates CNS prion disease and increases the accumulation of prions in the brain, suggesting that microglia provide neuroprotection by phagocytosing and destroying prions. In Csf1rΔFIRE mice, the deletion of an enhancer within Csf1r specifically blocks microglia development, however, their brains develop normally and show none of the deficits reported in other microglia-deficient models. Csf1rΔFIRE mice were used as a refined model in which to study the impact of microglia-deficiency on CNS prion disease. Although Csf1rΔFIRE mice succumbed to CNS prion disease much earlier than wild-type mice, the accumulation of prions in their brains was reduced. Instead, astrocytes displayed earlier, non-polarized reactive activation with enhancedphagocytosis of neuronal contents and unfolded protein responses. Our data suggest that rather than simply phagocytosing and destroying prions, the microglia instead provide host-protection during CNS prion disease and restrict the harmful activities of reactive astrocytes.