Role of CSF1/CSF1R signalling in avian macrophage biology

Abstract

The mononuclear phagocyte system (MPS), which is a heterogenous family of functionally related cells, includes myeloid progenitors, blood monocytes, resident tissue macrophages, bone osteoclasts and conventional dendritic cells. In mammals, macrophage colony stimulating factor (M-CSF or CSF1) promote differentiation, proliferation and survival of myeloid progenitor cells into mononuclear phagocyte lineage cells by binding and signalling activity through a surface receptor (CSF1R). Interleukin-34 or IL34 is alternative growth factor which also signals via CSF1R. CSF1, IL34 and the shared receptor CSF1R was shown to be conserved in birds, but their functions have not been studied in detail. The primary aim of this project is to study the role of CSF1R signalling in avian macrophage biology using three different approaches. The first approach involved the identification of chicken CSF1R specific kinase inhibitors, from a set of candidate mammalian CSF1R. Candidate CSF1R inhibitors were screened based on cell viability assay using IL-3 dependent pro B cell line Ba/F3 ectopically expressing chicken CSF1R and chicken bone marrow-derived macrophages (BMDM). To support these studies, biologically active, endotoxin-free recombinant chicken CSF1 protein was produced and refolded from inclusion bodies using a bacterial system. Out of 10 potential CSF1R inhibitors screened, 6 inhibitors TIA086, TIA02-052, TIA02-054, TIA02-076, KUL01-123 and KUL02- 016 were potent and selective for chicken CSF1R, whilst having no effect on growth in IL-3. Two inhibitors TIA02-054 and TIA02-076 were specific for the chicken CSF1R kinase compared to their actions on human CSF1R expressed in the same cells. The chicken CSF1R kinase inhibitors also effectively blocked CSF1-induced survival of primary BMDMs. BMDM survival was reduced even in the absence of exogenous CSF1 indicating a growth factor independent, autocrine CSF1/CSF1R signalling function in chicken macrophages. The second approach to study CSF1 biology in the development of chicken MPS involved use of a novel neutralising monoclonal antibody to chicken CSF1 (ROS-AV183) that targets and blocks chicken CSF1R signalling activity. In order to test the activity of anti-ChCSF1 mAb on chicken macrophages both in vitro and in vivo, both anti-ChCSF1 mAb and Isotype control mAb reagents were purified from hybridoma culture by affinity chromatography and characterized further for purity, size by SDS PAGE and CSF1R signaling blocking activity by BaF3/ChCSF1R cell viability assay. Anti-ChCSF1 mAb completely inhibited survival of primary chicken macrophages, irrespective of the presence or absence of CSF1, supporting the earlier finding regarding the autocrine CSF1 signalling behaviour of chicken macrophages. To determine the impact of anti-ChCSF1 mAb on postnatal birds in vivo, transgenic CSF1R-eGFP reporter birds were injected with antibody for four consecutive days. Anti-ChCSF1 mAb had no effect on the average growth rate, the relative weight gain or the normal development of hatchling birds. Anti-ChCSF1 mAb had no detectable effect on circulating CSF1 levels on the day of hatch or a week after treatment. Anti-ChCSF1 mAb significantly reduced CSF1R-eGFP transgene positive macrophages in bursa of Fabricius and caecal tonsil tissue, but not in spleen tissue. In bursa of Fabricius tissue, follicle associated epithelium (FAE) cell’s proliferation and survival was altered post treatment. In caecal tonsil anti-ChCSF1 mAb substantially reduced B lymphocytes; this depletion was also evident in the circulation and spleen tissue. Tissue resident MHC-II+ macrophages in spleen were effectively depleted, validating CSF1 dependency of tissue resident macrophages. In liver tissue, anti-ChCSF1 mAb treatment completely ablated Kupffer cell population. In bones anti-ChCSF1 mAb treatment depleted osteoclasts number. MicroCT scan analysis of bone femur architecture revealed significant reduction in the % bone volume and trabecular number, with a corresponding increase in the trabecular separation post anti-ChCSF1 mAb treatment of hatchling birds. In overview, the analysis indicated that CSF1 is required for post-hatch development of the MPS in birds and suggest trophic roles for CSF1-dependent macrophages in B cell development. The third approach involved deletion of CSF1R in the chicken genome using CRISPR Cas9 editing in chicken primordial germ cells (PGCs). Out of the several guide RNAs (gRNAs) designed targeting different regions of CSF1R loci, gRNAs targeting exon 1 and 10 (encoding transmembrane domain of the receptor) were functionally validated for mutation. Guide RNAs targeting exon 1 and transmembrane domain region were effective in mutating receptor CSF1R in cultured PGCs with targeting efficiency of around 35% and 100% respectively. Transplantation of PGCs with biallelic deleted transmembrane domain region of CSF1R into germ cell deficient chicken embryos gave rise to one founder female G0 bird containing edited donor PGCs. Breeding of this chicken upon sexual maturation with transgenic CSF1R-eGFP male established 30 CSF1R heterozygous G1 birds containing CSF1R edited donor PGCs (39% germline efficiency). CSF1R heterozygous G1 birds had no obvious phenotypes compared to wild type hatch mates throughout the development of embryos and in adults. Furthermore, CSF1R homozygous mutant embryos (G2) were generated by breeding CSF1R heterozygous G1 chickens (26% germline efficiency). Analysis of 8-day old CSF1R homozygous mutant embryos revealed deficiency in the expression of CSF1R protein in mononuclear phagocyte population. Hence, there was successful transmission of CSF1R knockout allele in G1 and G2 progeny. Analysis of the phenotype of the homozygous CSF1R mutant birds is ongoing. The novel tools characterized in this project, anti-ChCSF1 antibody, chicken CSF1R kinase domain inhibitors and CSF1R-deficient transgenic chicken line will enable further detailed studies of the role of macrophages in chicken immunity and development.