Explorer Pleiotropic effects of extended blockade of CSF 1 R 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 b 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. J. Leukoc. Biol. 96: 265–274; 2014. Introduction CSF1 controls the proliferation, differentiation, maturation, and survival of cells of the mononuclear phagocyte system [1–4]. The effects of CSF1 are mediated through the CSF1R, a protein tyrosine kinase receptor. Expression of Csf1r mRNA is myeloid-restricted in adult animals, and a Csf1r-EGFP reporter gene provides a convenient marker for macrophage-lineage cells in transgenic mice [5]. A natural mutation of the Csf1 gene in mice (op/op) produces a reduction in macrophage numbers in most tissues of the body, accompanied by severe growth retardation, osteopetrosis, and deficiencies in sensory, reproductive, and other endocrine systems [3, 4]. A null mutation of Csf1r produces even more penetrant phenotypes, including a significant postnatal mortality [6]. A second ligand for CSF1R, IL-34, provides an explanation for the greater impact of receptor depletion. IL-34 appears to be required for the generation of specific macrophage populations, notably microglia in the brain and epidermal Langerhans cells [7, 8]. CSF1 is not available in saturating concentrations in an adult mouse; the concentration is regulated, in part, by macrophagemediated clearance in the liver and spleen, providing a relatively simple homeostatic control of macrophage numbers [9]. Treatment of mice with CSF1 causes a massive increase in tissue macrophage numbers throughout the body. Surprisingly, this leads to a rapid increase in the size of the liver and hepatocyte proliferation, suggesting a function for CSF1-dependent macrophages in liver homeostasis (unpublished results). Conversely, continuous CSF1R signaling is required for the maintenance of macrophage populations in adult mice. The administration of a blocking mAb against the CSF1R gradually eliminated resident tissue macrophages from many different organs. The antibody did not prevent monocytopoiesis but apparently, prevented maturation of monocytes in peripheral blood to form the nonclassical Ly6C population [10, 11]. Consequently, the treatment did not prevent macrophage recruitment into inflammatory sites; indeed, the 1. Correspondence: The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Scotland EH25 9RG, UK. E-mail: david.hume@roslin.ed.ac.uk This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) (http://creativecommons. org/licenses/by/4.0/) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abbreviations: BV/TV 5 bone volume over total volume (bone density), CT 5 computed tomography, EGFP 5 enhanced green fluorescent protein, FSC 5 forward-scatter, IGF1 5 insulin-like growth factor 1, LH 5 luteinizing hormone, MicroCT 5 microcomputed tomography, OCL 5 osteoclasts, PAS 5 periodic acid—Schiff, PFA 5 paraformaldehyde, RBC 5 red blood cell, SSC 5 side-scatter, Tb.N 5 trabecular numbers, Tb.Th 5 trabecular thickness, TRAP 5 tartrate-resistant acid phosphatase, R(D)SVS 5 Royal (Dick) School of Veterinary Studies, WBC 5 white blood cell The online version of this paper, found at www.jleukbio.org, includes supplemental information. 0741-5400/14/0096-265 © The Author(s) Volume 96, August 2014 Journal of Leukocyte Biology 265 treatment exacerbated pathology in a model of graft-versushost disease. These basic findings were subsequently repeated using a different mAb [12]. The relatively slow depletion of tissue macrophages in the treated mice suggests that the major effect of anti-CSF1R is to prevent their replacement, via recruitment or local self-renewal. This view has been confirmed recently in models in which tissue macrophages have been acutely depleted, and replenishment is blocked by antiCSF1R [13]. The slow turnover of tissue macrophages means that after 3 weeks of treatment used previously, mice had only been fully depleted for a short period, and the consequences may not have become evident. Accordingly, in the current study, we aimed to define the impact of long-term treatment with anti-CSF1R. MATERIALS AND METHODS