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The meningeal space is a critical brain structure providing immunosurveillance for the central nervous system (CNS), 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, accumulates 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, by combining single-cell transcriptomics and mass cytometry by time-of-flight (CyTOF) with in vivo interventions, we found that chronic T. brucei infection triggers the development of ectopic lymphoid aggregates (ELAs) in the murine meninges. These infection-induced ELAs were defined by the presence of ER-TR7+ fibroblastic reticular cells, CD21/35+ follicular dendritic cells (FDCs), CXCR5+ PD1+ T follicular helper-like phenotype, GL7+ CD95+ GC-like B cells, and plasmablasts/plasma cells. Furthermore, the B cells found in the infected meninges produced high-affinity autoantibodies able to recognise mouse brain antigens, in a process dependent on LTβ signalling. A mid-throughput screening identified several host factors recognised by these autoantibodies, including myelin basic protein (MBP), coinciding with cortical demyelination and brain pathology. In humans, we identified the presence of autoreactive IgG antibodies in the cerebrospinal fluid (CSF) of second stage HAT patients that recognised human brain lysates and MBP, consistent with our findings in experimental infections. Lastly, we found that the pathological B cell responses we observed in the meninges required the presence of T. brucei in the CNS, as suramin treatment before the onset of the CNS stage prevented the accumulation of GL7+ CD95+ GC-like B cells and brain-specific autoantibody deposition. Taken together, our data provide evidence that the meningeal immune response during chronic T. brucei infection results in the acquisition of lymphoid tissue-like properties, 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, including neuropsychiatric lupus and multiple sclerosis.

Specific Escherichia coli isolates lysogenised with prophages that express Shiga toxin (Stx) can be a threat to human health, with cattle being an important natural reservoir. In many countries the most severe pathology is associated with enterohaemorrhagic E. coli (EHEC) serogroups that express Stx subtype 2a. In the United Kingdom, phage type (PT) 21/28 O157 strains have emerged as the predominant cause of life-threatening EHEC infections and this phage type commonly encodes both Stx2a and Stx2c toxin types. PT21/28 is also epidemiologically linked to super-shedding (>103 cfu/g of faeces) which is significant for inter-animal transmission and human infection as demonstrated using modelling studies. We demonstrate that Stx2a is the main toxin produced by stx2a+/stx2c+ PT21/28 strains induced with mitomycin C and this is associated with more rapid induction of gene expression from the Stx2a-encoding prophage compared to that from the Stx2c-encoding prophage. Bacterial supernatants containing either Stx2a and/or Stx2c were demonstrated to restrict growth of bovine gastrointestinal organoids with no restriction when toxin production was not induced or prevented by mutation. Isogenic strains that differed in their capacity to produce Stx2a were selected for experimental oral colonisation of calves to assess the significance of Stx2a for both super-shedding and transmission between animals. Restoration of Stx2a expression in a PT21/28 background significantly increased animal-to-animal transmission and the number of sentinel animals that became super-shedders. We propose that while both Stx2a and Stx2c can restrict regeneration of the epithelium, it is the relatively rapid and higher levels of Stx2a induction, compared to Stx2c, that have contributed to the successful emergence of Stx2a+ E. coli isolates in cattle in the last 40 years. We propose a model in which Stx2a enhances E. coli O157 colonisation of in-contact animals by restricting regeneration and turnover of the colonised gastrointestinal epithelium.

Many natural prion diseases of humans and animals are considered to be acquired through oral consumption of contaminated food or pasture. Determining the route by which prions establish host infection will identify the important factors that influence oral prion disease susceptibility and to which intervention strategies can be developed. After exposure, the early accumulation and replication of prions within small intestinal Peyer's patches is essential for the efficient spread of disease to the brain. To replicate within Peyer's patches, the prions must first cross the gut epithelium. M cells are specialised epithelial cells within the epithelia covering Peyer's patches that transcytose particulate antigens and microorganisms. M cell-development is dependent upon RANKL-RANK-signalling, and mice in which RANK is deleted only in the gut epithelium completely lack M cells. In the specific absence of M cells in these mice, the accumulation of prions within Peyer's patches and the spread of disease to the brain was blocked, demonstrating a critical role for M cells in the initial transfer of prions across the gut epithelium in order to establish host infection. Since pathogens, inflammatory stimuli and aging can modify M cell-density in the gut, these factors may also influence oral prion disease susceptibility. Mice were therefore treated with RANKL to enhance M cell density in the gut. We show that prion uptake from the gut lumen was enhanced in RANKL-treated mice, resulting in shortened survival times and increased disease susceptibility, equivalent to a 10-fold higher infectious titre of prions. Together these data demonstrate that M cells are the critical gatekeepers of oral prion infection, whose density in the gut epithelium directly limits or enhances disease susceptibility. Our data suggest that factors which alter M cell-density in the gut epithelium may be important risk factors which influence host susceptibility to orally acquired prion diseases.

The MacBlue transgenic mouse uses the Csf1r promoter and first intron to drive expression of gal4-VP16, which in turn drives a cointegrated gal4-responsive UAS-ECFP cassette. The Csf1r promoter region used contains a deletion of a 150 bp conserved region covering trophoblast and osteoclast-specific transcription start sites. In this study, we examined expression of the transgene in embryos and adult mice. In embryos, ECFP was expressed in the large majority of macrophages derived from the yolk sac, and as the liver became a major site of monocytopoiesis. In adults, ECFP was detected at high levels in both Ly6C+ and Ly6C- monocytes and distinguished them from Ly6C+, F4/80+, CSF1R+ immature myeloid cells in peripheral blood. ECFP was also detected in the large majority of microglia and Langerhans cells. However, expression was lost from the majority of tissue macrophages, including Kupffer cells in the liver and F4/80+ macrophages of the lung, kidney, spleen and intestine. The small numbers of positive cells isolated from the liver resembled blood monocytes. In the gut, ECFP+ cells were identified primarily as classical dendritic cells or blood monocytes in disaggregated cell preparations. Immunohistochemistry showed large numbers of ECFP+ cells in the Peyer's patch and isolated lymphoid follicles. The MacBlue transgene was used to investigate the effect of treatment with CSF1-Fc, a form of the growth factor with longer half-life and efficacy. CSF1-Fc massively expanded both the immature myeloid cell (ECFP-) and Ly6C+ monocyte populations, but had a smaller effect on Ly6C- monocytes. There were proportional increases in ECFP+ cells detected in lung and liver, consistent with monocyte infiltration, but no generation of ECFP+ Kupffer cells. In the gut, there was selective infiltration of large numbers of cells into the lamina propria and Peyer's patches. We discuss the use of the MacBlue transgene as a marker of monocyte/macrophage/dendritic cell differentiation.

Prion diseases are characterised by the accumulation of PrP(Sc), an abnormally folded isoform of the cellular prion protein (PrP(C)), in affected tissues. Following peripheral exposure high levels of prion-specific PrP(Sc) accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrP(C) is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrP(C) expression was specifically "switched on" or "off" only on FDC. We show that PrP(C)-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrP(C)-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrP(C) expression on FDC.