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CSF1R-dependent macrophages control postnatal somatic growth and organ maturation

['Sahar Keshvari', 'Mater Research Institute-University Of Queensland', 'Translational Research Institute', 'Woolloongabba', 'Brisbane', 'Qld', 'Melanie Caruso', 'Ngari Teakle', 'Lena Batoon', 'Anuj Sehgal']

Date: None

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43 hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.

Monocytes and macrophages are cells of the innate immune system produced by the bone marrow that can be recruited into tissues to support defense against infection and repair following injury. So-called resident macrophages are abundant in every tissue in the body. Their numbers are controlled by a hormone-like growth factor called macrophage colony-stimulating factor (CSF1). Mutations in the surface receptor (CSF1R) that enables macrophages to respond to CSF1 lead to the loss of tissue macrophages. Human patients born with CSF1R mutations may die in infancy or suffer severe developmental abnormalities in the skeleton and brain. In this study we report the effects of the loss of tissue macrophages on postnatal growth and development in a rat model of CSF1R deficiency. In this model there was a global loss of resident macrophages, severe postnatal growth defects, failure of development of multiple organs including the liver and early death. The effects of the mutation could be rescued completely by transfer of normal bone marrow cells into the peritoneal cavity at weaning permitting long term survival and even fertility. The results indicate that postnatal expansion of tissue macrophage populations is essential for normal development.

Funding: The generation of the Csf1rko rat was supported by UK Medical Research Council Grant MR/M019969/1 to DAH and CP. This work was supported in part by Australian National Health and Medical Research Council (NHMRC) Grant GNT1163981 awarded to DAH and KMS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2021 Keshvari et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Phenotypes associated with biallelic recessive mutations in human CSF1R appear somewhat less severe although it is not clear that any such mutations are definitively null for CSF1R function (reviewed in [ 6 ]). Patients present with abnormal skeletal development and calcification, ventricular enlargement (hydrocephalus) and selective loss of microglia in the brain leading to degenerative encephalopathy and brain malformations [ 8 – 11 ]. Although utility is compromised by the comparative lack of reagents, the rat has many advantages over the mouse for the study of development, physiology, pathology and mononuclear phagocyte homeostasis (reviewed in [ 12 ]). We previously generated and characterised Csf1rko rats as an alternative model of human CSF1R deficiency [ 13 ]. This model was initially established and analysed on a mixed genetic background to avoid the pre-weaning mortality seen in Csf1rko mice. Although the gross osteopetrotic phenotype of these rats was 100% penetrant, post-weaning survival was variable and apparently female-biased [ 13 ]. To enable us to test complementation of the Csf1rko mutation by transfer of bone marrow (BM) and to establish a more consistent model we back-crossed the original outbred line to the dark agouti (DA) inbred background, which was the origin of the ES cells used in homologous recombination to generate the original Csf1rko rats [ 13 ]. Unlike inbred Csf1rko mice, the majority of the inbred DA Csf1rko rats survive to adulthood. In the present study we use the inbred line to analyse the profound impacts of the Csf1rko on tissue monocyte and macrophage populations and the growth and development of the skeleton and major organs in the early postnatal period. We show that CSF1R-dependent macrophages are essential in the liver for postnatal hyperplasia, hepatocyte functional maturation and lipid metabolism and that resident macrophage populations and the pleiotropic Csf1rko phenotypes can be almost completely reversed by transfer of BM cells from wild type (WT) congenic animals at weaning. Our findings demonstrate that expansion and maturation of resident tissue macrophage populations is a key event in postnatal development that controls somatic growth and organ development.

Resident macrophages are abundant in every tissue in the body and adapt to each tissue environment by expressing unique gene sets required for their local functions (reviewed in [ 1 , 2 ]). Their differentiation from progenitor cells, their gene expression profile and their survival/maintenance in tissues is controlled by two ligands, colony stimulating factor 1 (CSF1) and interleukin 34 (IL34), which each signal through the CSF1 receptor (CSF1R) (reviewed in [ 3 ]). The biology of CSF1R and its ligands is conserved from fish and birds to mammals [ 4 – 6 ]. In mice, the impacts of a homozygous Csf1r knockout mutation (Csf1rko) include perinatal mortality, postnatal growth retardation, increased bone density (osteopetrosis), global defects in brain development and abnormalities of the sensory nervous system, infertility and delayed pancreatic beta cell development (reviewed in [ 3 , 7 ]). Many of the effects of the Csf1rko in mice are shared with mutations in the Csf1 gene [ 3 , 7 ].

Results

The effect of the Csf1rko on skeletal development The skeletal phenotype of the outbred adult Csf1rko rat [13] closely resembles that of human CSF1R-deficient patients, referred to as dysosteosclerosis [8]. Dai et al. [19] reported disorganized matrix, reduced mineralization and osteoblast deficiency in the bones of 2–3 wk old Csf1rko mice. As shown in Fig 3A, analysis of the juvenile inbred Csf1rko rats revealed that a delay in skeletal calcification was already evident in newborns. By 3 wks of age secondary ossification centers of long bones and the small bones in the fore and hind paws were clearly deficient (Fig 3B and 3C). Unlike the hyper-calcified skull base [13], a shared feature with human bi-allelic CSF1R mutation [8,10,20], the cranial case of the Csf1rko rats remained hypomineralized even in adults and closure of the sutures was impaired (Fig 3D). The delay in postnatal musculoskeletal development was reflected in muscle. Sections through muscle at 7 days, 3 wks and 7 wks stained with laminin (Fig 3E and 3F) demonstrate that the relative cellularity was similar and the reduced muscle mass in the Csf1rko was primarily associated with a reduction in muscle fibre diameter (i.e. failure of hypertrophy). PPT PowerPoint slide

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larger image TIFF original image Download: Fig 3. Postnatal skeletal development in Csf1rko rats. (A) Representative 3D reconstruction of micro-CT images in newborn (Week 0) and 1wk old WT and Csf1rko rats. Digits of forepaws in white boxes. (B) Alcian blue and Alizarin red staining of newborn WT and Csf1rko rats with ossified cartilage/bone stained red (black arrows) and unossified cartilage/bone stained blue. (C) Representative 3D reconstructed micro-CT images of 3-week old rat ribcage (left panel) and hind paw (right panel) with arrow heads indicating variance in ossification in distal metatarsal/proximal phalanges. (D) Representative 3D reconstructed micro-CT images of 7-wk old rat skull of WT (top) and Csf1rko (bottom) rats. (E) Representative images of laminin staining of hindlimb muscles of WT and Csf1rko rats at 1, 3 and 7 wks. (F) Average diameter of muscle fibres from the posterior tibialis in WT and Csf1rko rats at 3 wks. Original magnification: 400X. Scale bar: 20 μm. https://doi.org/10.1371/journal.pgen.1009605.g003

The effect of the Csf1rko on major organs Further analysis of juvenile rats indicated that the developmental delay in the inbred Csf1rko was not confined to the liver. One phenotype we did not note previously was almost complete involution of the thymus which was almost undetectable by 7 weeks of age (not shown). A subset of major organs is shown in S2 Fig. In the rat, the process of nephrogenesis continues in the immediate postnatal period [30]; the impact of the Csf1rko on the kidney was not previously analysed [13]. By 7 wks of age, we observed profound renal medullary hypoplasia, which was so severe that in most Csf1rko rats examined there was just a bud of papilla within the central pelvicalyceal space. The cortex was correspondingly hyperplastic, but the tubules and glomeruli appeared relatively normal (S2A and S2B Fig). The intestines of outbred adult Csf1rko rats were not grossly abnormal and by contrast to Csf1op/op mice, they were not Paneth cell deficient [13]. To dissect more subtle impacts of the Csf1rko in the inbred line we performed quantitative analysis of villus architecture in the ileum. There were significant reductions in the length and width of the crypts and villi, and the thickness of the submucosa in the Csf1rko (S2C–S2F Fig). Interestingly, given the reported role of CSF1R dependent macrophages in M cell differentiation in mice [31], we noted for the first time that macroscopic Peyer’s patches were almost undetectable in the Csf1rko rats at 3 wks and remained so at later ages. The main welfare concern with the inbred Csf1rko is the development of progressive breathing difficulty. We considered the possibility that macrophage deficiency might also impact postnatal development of the lung. S2G Fig shows images of the inflated lungs of WT and Csf1rko rats at 3 wks stained with aldehyde-fuchsin to highlight elastin fibres and S2H Fig quantitates the airway space. There was no unequivocal evidence of impaired alveolisation. Flow cytometric analysis of disaggregated lung tissue revealed an increase in granulocytes in Csf1rko rats by 9 wks (S2I and S1J Figs), as seen in peripheral blood and BM, but there remained no histological evidence of inflammation that could explain the impaired respiratory function. As in the outbred line [13], male and female inbred Csf1rko rats lacked development of primary and secondary reproductive organs. In males, the prostate and seminal vesicles and in females the uterus were so under-developed as to be almost undetectable (not shown). CSF1R-dependent macrophages have also been implicated in the process of branching morphogenesis in the mammary gland in mice [32]. This was not previously analysed in the rat. S3 Fig shows a comparison of mammary gland development in Csf1rko and WT female DA rats at around 9 wks. The negative impact of the mutation on ductal development was evident from staining with mature epithelial cell markers (KRT5, E-cadherin).

The effect of the Csf1rko on tissue macrophages Aside from limited analysis using CD68 as a marker in adult liver and spleen, the effect of the Csf1rko on resident macrophages other than microglia was not examined previously [13]. To visualise resident macrophages we crossed the Csf1rko back to the Csf1r-mApple reporter transgene on the outbred SD background [33]. Fig 6 shows detection of the mApple transgene in a diverse set of tissues from WT and Csf1rko rats on this genetic background at weaning. In most tissues there was complete loss of Csf1r-mApple expressing cells aside from occasional monocyte-like cells and granulocytes in the vessels. This includes the abundant resident macrophage populations in smooth and skeletal muscle, kidney, pancreas, adipose, salivary and adrenal glands that were not recognised or analysed previously in mice. The pancreas contains numerous small lymph nodes that contain abundant Csf1r-mApple-positive cells. Whereas peri-acinar and islet macrophage populations were entirely lost in the Csf1rko, the lymph node-associated populations were partly retained and highlighted in whole mounts (Fig 6D). Partial reductions in resident populations were observed in intestinal mucosa, liver and lung (Fig 6C, 6H and 6J) Note that the resident Csf1r-mApple expressing cells in non-lymphoid tissues that are missing in the Csf11rko would include populations classified as dendritic cells, which are also CSF1R-dependent in mice [34]. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 6. The effect of the Csf1rko on distribution of macrophages detected using a Csf1r-mApple reporter gene. Tissues were extracted from 3 week old rats generated by crossing heterozygous inbred DA Csf1rko rats with outbred (SD) Csf1r-mApple reporter rats and then inter-breeding selected progeny to generate WT and mutant rats also expressing the mApple reporter. Images are maximum intensity projection of z-stack series of whole-mounted (A) colon muscularis (smooth muscle), (B) skeletal muscle, (C) colonic mucosa, (D) pancreas, (E) perigonadal white adipose tissue, (F) salivary glands, (G) adrenal glands, (H) liver, (I) kidney cortex and (J) lung. Scale bars = 200um. https://doi.org/10.1371/journal.pgen.1009605.g006

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