Transcriptome of microglia reveals a species‐specific expression profile in bovines with conserved and new signature genes

Abstract

Evidence is growing that microglia adopt different roles than monocyte‐derived macrophages (MDM) during CNS injury. However, knowledge about their function in the pathogenesis of neuroinfections is only rudimentary. Cattle are frequently affected by neuroinfections that are either zoonotic or related to diseases in humans, and, hence, studies of bovine neuroinfections as a natural disease model may generate fundamental data on their pathogenesis potentially translatable to humans. We investigated the transcriptomic landscape and lineage markers of bovine microglia and MDM. Although bovine microglia expressed most microglial signature genes known from humans and mice, they exhibited a species‐specific transcriptomic profile, including strikingly low expression of TMEM119 and enrichment of the two scavenger receptors MEGF10 and LY75. P2RY12 was amongst the most enriched genes in bovine microglia, and antibodies against P2RY12 labeled specifically resting microglia, but also reactive microglia within neuroinfection foci in‐situ. On the other hand, F13A1 was amongst the most enriched genes in bovine monocytes and MDM and, additionally, the encoded protein was expressed in‐situ in monocytes and MDM in the inflamed brain but not in microglia, making it a promising marker for infiltrating MDM in the brain. In culture, primary bovine microglia downregulated signature genes, expressed markers of activation, and converged their transcriptome to MDM. However, they retained several microglia signature genes that clearly distinguished them from bovine MDM, making them a promising in‐vitro tool to study mechanisms of microglia–pathogen interactions.

Bovine neutrophil chemotaxis to Listeria monocytogenes in neurolisteriosis depends on microglia-released rather than bacterial factors

“…Interestingly, supernatant from non-infected microglia could also elicit modest PMN chemotaxis, albeit at significantly lower levels than Lm -infected microglia supernatant (Fig. 3 D), giving further support to transcriptomic data indicating activation of bovine microglia following isolation and cell culture [ 42 ]. Taken together, these data indicate that Lm -infected microglia release factors chemotactic for bovine PMN, suggesting that microglial cells are the major players in PMN recruitment to infectious foci during neurolisteriosis.…”

“…Similar to previously reported data [ 63 ], the vast majority of cultured cells stained for GFAP (95.53% ± 2.37 SD), confirming their astrocytic lineage, and Vimentin (98.56% ± 0.93 SD), with only few Iba1-positive cells (0.68% ± 0.51 SD) identifiable as contaminating microglia. For primary microglial cell cultures, and as previously characterized [ 42 ], approximately 100% of cells expressed Iba1 (not shown) indicating high microglial purity.…”

“…No. P1644, Sigma) gradient centrifugation, and were cultured as recently described [ 42 ]. As assessed through recently described characterization methods [ 42 ], almost pure bovine microglial cultures were obtained.…”

“…For both IHC and IF, dewaxing, antigen retrieval (citrate buffer, pH 6) and blocking with 5% normal goat serum (NGS) were performed as previously described [ 42 ].…”

“…For IF, incubation with rabbit anti-P2RY12 (1:200, 55043A, AnaSpec), rabbit anti-Iba1 (1:500, 013-27593, Wako) and/or mouse Factor XIII A (F13A1, 1:100, MA5-11751, Thermo Fisher) were performed as previously described [ 42 ]. In addition, sections were incubated with mouse anti-sheep IL-8 antibody (1:400, MCA1660, clone 8M6, Bio-Rad) overnight at 4 °C, following antigen retrieval in Target Retrieval solution, pH 9 (S2367, Agilent Technologies) for 15 min at 95 °C in a H2850 Microwave Processor (EBSciences, East Granby, Hartford County, Connecticut).…”