Antigen Sampling CSF1R-Expressing Epithelial Cells Are the Functional Equivalents of Mammalian M Cells in the Avian Follicle-Associated Epithelium

Abstract

The follicle-associated epithelium (FAE) is a specialized structure that samples luminal antigens and transports them into mucosa-associated lymphoid tissues (MALT). In mammals, transcytosis of antigens across the gut epithelium is performed by a subset of FAE cells known as M cells. Here we show that colony-stimulating factor 1 receptor (CSF1R) is expressed by a subset of cells in the avian bursa of Fabricius FAE. Expression was initially detected using a CSF1R-reporter transgene that also label subsets of bursal macrophages. Immunohistochemical detection using a specific monoclonal antibody confirmed abundant expression of CSF1R on the basolateral membrane of FAE cells. CSF1R-transgene expressing bursal FAE cells were enriched for expression of markers previously reported as putative M cell markers, including annexin A10 and CD44. They were further distinguished from a population of CSF1R-transgene negative epithelial cells within FAE by high apical F-actin expression and differential staining with the lectins jacalin, PHA-L and SNA. Bursal FAE cells that express the CSF1R-reporter transgene were responsible for the bulk of FAE transcytosis of labeled microparticles in the size range 0.02–0.1 μm. Unlike mammalian M cells, they did not readily take up larger bacterial sized microparticles (0.5 μm). Their role in uptake of bacteria was tested using Salmonella, which can enter via M cells in mammals. Labeled Salmonella enterica serovar Typhimurium entered bursal tissue via the FAE. Entry was partially dependent upon Type III secretion system-1. However, the majority of invading bacteria were localized to CSF1R-negative FAE cells and in resident phagocytes that express the phosphatidylserine receptor TIM4. CSF1R-expressing FAE cells in infected follicles showed evidence of cell death and shedding into the bursal lumen. In mammals, CSF1R expression in the gut is restricted to macrophages which only indirectly control M cell differentiation. The novel expression of CSF1R in birds suggests that these functional equivalents to mammalian M cells may have different ontological origins and their development and function are likely to be regulated by different growth factors.

Inside-out chicken enteroids with leukocyte component as a model to study host–pathogen interactions

“…Salmonella enterica subspecies enterica serovar Typhimurium strain 4/74 carrying a chromosomal pFVP25.1::gfp fusion linked to the naladixic acid resistance gene was utilised for infections of the chicken enteroids and compared to a defined mutant, ST4/74 nal R ΔprgH::kan (kindly provided by Dr P. Vohra). This prgH mutant is confirmed to have reduced Type 3 secretion by analysing secretion of SipC, a Salmonella type III secretion system (T3SS) effector protein, and was also transformed with the plasmid pFVP25.1 which constitutively expresses GFP 67 , 68 . Strains were cultured overnight in Luria-Bertani (LB) broth with 50 µg/mL kanamycin (not used for wild-type ST4/74 nal R ), 50 µg/mL ampicillin and 20 µg/mL naladixic acid at 37 °C.…”

Development of novel reagents to chicken FLT3, XCR1 and CSF2R for the identification and characterization of avian conventional dendritic cells

“…To differentiate phagocytosis from active invasion of cells by Salmonella Typhimurium, CSF1R-eGFP + splenic MPS cells were exposed in vitro to either wild-type (WT) or a non-invasive ΔprgH mutant S. Typhimurium at a multiplicity of infection of five. Both bacterial strains expressed the fluorescent protein mCherry [32]. The cDC and macrophages were gated based on levels of FLT3 expression.…”

A Transgenic Line That Reports CSF1R Protein Expression Provides a Definitive Marker for the Mouse Mononuclear Phagocyte System

“…Deletion of a conserved enhancer in the first intron (Fms intronic regulatory element, FIRE) leads to selective loss of Csf1r expression in tissue macrophage populations including microglia in the brain (4). Transgenic reporters containing FIRE have been generated in mice (5,6) rats (7), chickens (8,9) and sheep (10) and in each species highlight the location and abundance of macrophage populations in every organ.…”