Authors

R Heslop*¹; C Bentley-Abbot*¹; P Chandrasegaran¹; M Sinton¹; A MacLeod¹; JF Quintana¹;  ¹ Wellcome Centre for Integrative Parasitology (WCIP), UK

Discussion

Inflammation is an essential protective response against invading pathogens, but its resolution is required to limit host tissue damage. Regulatory B cells (Bregs) play a critical role in inflammation resolution by the release of anti-inflammatory cytokines IL-10, TGFβ and IL-35. However, the molecular and cellular mechanisms by which Bregs contribute to the anti-inflammatory response in the brain is understudied. Trypanasoma brucei  invades the CNS during chronic infection, causing gliosis and neuroinflammation. Using spatial and single cell transcriptomics, we have recently identified a bidirectional crosstalk between brain dwelling Bregs and microglia mediated by the anti-inflammatory cytokine Il10 and the survival factor Tnfsf13b. Here, we validated the transcriptional profiles of these cells in the circumventricular organs (CVOs) of T. brucei infected mice using single molecule fluorescent in situ hybridisation (smFISH). We have developed a robust analytical pipeline using Qupath to count cells positive for multiple targets with >75% accuracy. We are able to quantify single sub-cellular “transcriptional spots”, representing individual RNA transcripts, and clustering behaviour in host cells in a semi-automated manner and automatically partition cells by RNA expression. Furthermore, large datasets comprising multiple tissue sections can be analysed holistically and z-stacks reconstructed. Our pipeline accurately detected an increase in Il10 expression and in Cd79a+/Il10+ Bregs in the choroid plexus of infected mice compared to naïve controls, validating our multi-omics findings. We also visualised, for the first time, Gapdh+/Pyk1+ slender forms and Pad2+/Epi1+ stumpy forms of T. brucei in the choroid plexus of chronically infected mice using smFISH. Our pipeline offers efficient, automated data processing for multi-dimensional, multi-target QuPath generated smFISH datasets and can therefore be readily applied to a variety of biological questions. Integration of smFISH with this pipeline presents a means to investigate the molecular interactions between Bregs and microglia, or indeed other cell populations, in chronic CNS infection.