Role of macrophage colony stimulating factor-1 (CSF-1) in postnatal growth

Colony-Stimulating Factor (CSF-1) is required for the proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Mice with a mutation in their CSF-1 gene demonstrate abnormal development in many organ systems and severe growth retardation. These defects can be corrected by administration of rh- CSF-1, and when similarly administered to wild-type mice, can increase organ and body weight, thus highlighting the importance of CSF-1 in postnatal growth. CSF-1 is known to be elevated in the circulation in the immediate postnatal period of both mice and humans. It remains to be seen whether CSF-1 deficiency underlies important clinical issues such as low birth weight, and whether there are any functionally important variations in expression or biology of CSF-1, or the alternative CSF-1R ligand IL-34 that contributes to variation in somatic growth between individuals. This thesis aimed to use the pig as a model for human innate immunity and disease based upon recent publications that highlighted the similarities in their immune systems. To investigate the effects of CSF-1 on postnatal growth the first aim was to characterise the CSF-1R system in pigs and produce reagents. Biologically active porcine CSF-1 and IL-34 were produced along with expression of full length functional porcine CSF-1R and production of anti-CSF-1R antibodies. A bioassay was developed and optimised to assess the biological activity of these proteins. The cross-species reactivity of a range of species CSF-1 and IL-34 proteins was investigated in-vitro using the bioassay and cell culture systems. Recombinant CSF-1 is known to have a short half-life. Since conjugation of proteins to the Fc region of immunoglobulins has been used extensively to improve circulating half-life; a porcine Fc CSF-1 fusion protein was generated by commercial partners, Pfizer Animal Health. The conjugated and un-conjugated CSF-1 proteins had identical activity in cell line and primary cell assays in-vitro. The in-vivo activity of porcine Fc CSF-1 was tested initially in the Csf1r-EGFP+ mouse reporter line and C57BL/6 mice. The Fc CSF-1 protein was more active than the native protein in promoting increased monocyte and tissue macrophage numbers, increasing body weight and inducing hepatosplenomegaly. Hepatic growth was associated with extensive macrophage infiltration and hepatocyte proliferation, identified by gene expression profiling as well as immunohistochemistry. Fc CSF-1 was then tested in neonatal pigs. They were found to have an immature immune system that develops with age. No postnatal surge of CSF-1 was detected. Fc CSF-1 administration increased blood monocyte and neutrophil numbers confirming that CSF-1 is not saturating at this time. Nevertheless, no influence on postnatal growth rate was identified. This is discussed in terms of the differences in placental architecture in the pig compared to human and mouse. This thesis demonstrates the effectiveness of porcine Fc CSF-1 in both mice and porcine and highlights the important role that CSF-1 and macrophages play in liver homeostasis. Fc CSF-1 is identified as candidate therapeutic agent in humans and companion animals for tissue regeneration, and a tool for the study of the role of macrophages in physiology and pathology

Cloning and expression of feline colony stimulating factor receptor (CSF-1R) and analysis of the species specificity of stimulation by colony stimulating factor-1 (CSF-1) and interleukin-34 (IL-34).

AbstractColony stimulating factor (CSF-1) and its receptor, CSF-1R, have been previously well studied in humans and rodents to dissect the role they play in development of cells of the mononuclear phagocyte system. A second ligand for the CSF-1R, IL-34 has been described in several species. In this study, we have cloned and expressed the feline CSF-1R and examined the responsiveness to CSF-1 and IL-34 from a range of species. The results indicate that pig and human CSF-1 and human IL-34 are equally effective in cats, where both mouse CSF-1 and IL-34 are significantly less active. Recombinant human CSF-1 can be used to generate populations of feline bone marrow and monocyte derived macrophages that can be used to further dissect macrophage-specific gene expression in this species, and to compare it to data derived from mouse, human and pig. These results set the scene for therapeutic use of CSF-1 and IL-34 in cats

Cytology, culture and genomics to evaluate the microbiome in healthy rabbit external ear canals

Background: Lop-eared rabbits may be predisposed to otitis externa (OE) as a consequence of their ear conformation. Although otoscopy, otic cytological evaluation and culture are valuable tools in dogs and cats, published data on rabbits remain lacking. Hypothesis/Objectives: This study aimed to assess the utility of otoscopy and cytological results in evaluating healthy rabbit external ear canals (EECs) and to characterise ear cytological and microbiological findings through culture techniques and metagenomic sequencing. Animals: Sixty-three otitis-free client-owned rabbits. Materials and Methods: All rabbits underwent otoscopy and ear cytological evaluation. In a subset of 12 rabbits, further bacterial and fungal culture, fungal DNA assessment and metagenomic sequencing were performed. Results: Otic cytological results revealed yeast in 73%, cocci in 42.9% and rods in 28.6% of healthy rabbit EECs. Compared to upright-eared rabbits, lop-eared rabbits had more discharge and more bacteria per oil immersion field. Culture isolated eight different species yet metagenomic sequencing identified 36, belonging to the Bacillota (Firmicutes), Pseudomonadota and Actinomycetota phyla. Staphylococcus were the most commonly observed species with both methods. Ten of 12 rabbits were yeast-positive on cytological evaluation with only three yielding fungal growth identified as Yarrowia (Candida) lipolytica, Eurotium echinulatum and Cystofilobasidium infirmominiatum. Conclusions and Clinical Relevance: Healthy rabbit EECs lack inflammatory cells yet can host yeast and bacteria, emphasising the need to evaluate cytological results alongside the clinical signs. Lop-ear anatomy may predispose to bacterial overgrowth and OE. Notably, yeasts may be present despite a negative culture.</p

Mechanisms underlying the exquisite sensitivity of Candida albicans to combinatorial cationic and oxidative stress that enhances the potent fungicidal activity of phagocytes

Copyright © 2014 Kaloriti et al.Peer reviewedPublisher PD

The PTPN22 Locus and Rheumatoid Arthritis: No Evidence for an Effect on Risk Independent of Arg620Trp

The Trp(620) allotype of PTPN22 confers susceptibility to rheumatoid arthritis (RA) and certain other classical autoimmune diseases. There has been a report of other variants within the PTPN22 locus that alter risk of RA; protective haplotype '5', haplotype group '6-10' and susceptibility haplotype '4', suggesting the possibility of other PTPN22 variants involved in the pathogenesis of RA independent of R620W (rs2476601). Our aim was to further investigate this possibility.A total of 4,460 RA cases and 4,481 controls, all European, were analysed. Single nucleotide polymorphisms rs3789607, rs12144309, rs3811021 and rs12566340 were genotyped over New Zealand (NZ) and UK samples. Publically-available Wellcome Trust Case Control Consortium (WTCCC) genotype data were used.The protective effect of haplotype 5 was confirmed (rs3789607; (OR = 0.91, P = 0.016), and a second protective effect (possibly of haplotype 6) was observed (rs12144309; OR = 0.90, P = 0.021). The previously reported susceptibility effect of haplotype 4 was not replicated; instead a protective effect was observed (rs3811021; OR = 0.85, P = 1.4×10(-5)). Haplotypes defined by rs3789607, rs12144309 and rs3811021 coalesced with the major allele of rs12566340 within the adjacent BFK (B-cell lymphoma 2 (BCL2) family kin) gene. We, therefore, tested rs12566340 for association with RA conditional on rs2476601; there was no evidence for an independent effect at rs12566340 (P = 0.76). Similarly, there was no evidence for an independent effect at rs12566340 in type 1 diabetes (P = 0.85).We have no evidence for a common variant additional to rs2476601 within the PTPN22 locus that influences the risk of RA. Arg620Trp is almost certainly the single common causal variant

Pleiotropic effects of extended blockade of CSF1R signaling in adult mice.

We investigated the role of CSF1R signaling in adult mice using prolonged treatment with anti-CSF1R antibody. Mutation of the CSF1 gene in the op/op mouse produces numerous developmental abnormalities. Mutation of the CSF1R has an even more penetrant phenotype, including perinatal lethality, because of the existence of a second ligand, IL-34. These effects on development provide limited insight into functions of CSF1R signaling in adult homeostasis. The carcass weight and weight of several organs (spleen, kidney, and liver) were reduced in the treated mice, but overall body weight gain was increased. Despite the complete loss of Kupffer cells, there was no effect on liver gene expression. The treatment ablated OCL, increased bone density and trabecular volume, and prevented the decline in bone mass seen in female mice with age. The op/op mouse has a deficiency in pancreatic β cells and in Paneth cells in the gut wall. Only the latter was reproduced by the antibody treatment and was associated with increased goblet cell number but no change in villus architecture. Male op/op mice are infertile as a result of testosterone insufficiency. Anti-CSF1R treatment ablated interstitial macrophages in the testis, but there was no sustained effect on testosterone or LH. The results indicate an ongoing requirement for CSF1R signaling in macrophage and OCL homeostasis but indicate that most effects of CSF1 and CSF1R mutations are due to effects on development

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Characterisation of a novel Fc conjugate of Macrophage Colony-Stimulating Factor (CSF1)

We have produced an Fc conjugate of colony-stimulating factor (CSF) 1 with an improved circulating half-life. CSF1-Fc retained its macrophage growth-promoting activity, and did not induce proinflammatory cytokines in vitro. Treatment with CSF1-Fc did not produce adverse effects in mice or pigs. The impact of CSF1-Fc was examined using the Csf1r-enhanced green fluorescent protein (EGFP) reporter gene in MacGreen mice. Administration of CSF1-Fc to mice drove extensive infiltration of all tissues by Csf1r-EGFP positive macrophages. The main consequence was hepatosplenomegaly, associated with proliferation of hepatocytes. Expression profiles of the liver indicated that infiltrating macrophages produced candidate mediators of hepatocyte proliferation including urokinase, tumor necrosis factor, and interleukin 6. CSF1-Fc also promoted osteoclastogenesis and produced pleiotropic effects on other organ systems, notably the testis, where CSF1-dependent macrophages have been implicated in homeostasis. However, it did not affect other putative CSF1 targets, notably intestine, where Paneth cell numbers and villus architecture were unchanged. CSF1 has therapeutic potential in regenerative medicine in multiple organs. We suggest that the CSF1-Fc conjugate retains this potential, and may permit daily delivery by injection rather than continuous infusion required for the core molecule