Towards understandingTissue Mϕ heterogeneity and functions using quantitative proteomics
ABSTRACT: Resident macrophages are important for maintaining tissue homeostasis and for defence against infections, but their precise functions in different tissues are not fully elucidated.We have used high resolution quantitative proteomics to investigate the functions of splenic red pulp macrophages and peritoneal cavity macrophages in the steady-state. The validation of several proteins at cellular expression levels by the flow cytometry analysis was consistently in agreement with the proteomics data. Peritoneal macrophages were shown to be enriched in a number of key enzymes and metabolic pathways normally associated with the liver, such as metabolism of fructose, detoxification, nitrogen homeostasis and the urea cycle. Supporting observations in proteomics, we find that PM are able to utilise glutamine and glutamate which are rich in peritoneum for urea generation. In comparison, splenic red pulp macrophages were enriched in proteins important for adaptive immunity such as antigen presenting MHC molecules, in addition to proteins required for erythrocyte homeostasis and iron turnover. We also show that these tissue macrophages may utilise carbon and nitrogen substrates for different metabolic fates to support distinct tissue-specific roles. This study provides a valuable resource for biologists interested in the functions of tissue macrophages.
Project description:We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.
Project description:Tissue-resident macrophages in the spleen play a major role in the clearance of immunoglobulin G (IgG)-opsonized blood cells, as occurs in immune thrombocytopenia (ITP) and autoimmune hemolytic anemia (AIHA). Blood cells are phagocytosed via the Fc-? receptors (Fc?Rs), but little is known about the Fc?R expression on splenic red pulp macrophages in humans, with only a few previous studies that showed conflicting results. We developed a novel method to specifically isolate red pulp macrophages from 82 human spleens. Surface expression of various receptors and phagocytic capacity was analyzed by flow cytometry and immunofluorescence of tissue sections. Red pulp macrophages were distinct from splenic monocytes and blood monocyte-derived macrophages on various surface markers. Human red pulp macrophages predominantly expressed the low-affinity receptors Fc?RIIa and Fc?RIIIa. In contrast to blood monocyte-derived macrophages, red pulp macrophages did not express the inhibitory Fc?RIIb. Red pulp macrophages expressed very low levels of the high-affinity receptor Fc?RI. Messenger RNA transcript analysis confirmed this expression pattern. Unexpectedly and despite these differences in Fc?R expression, phagocytosis of IgG-opsonized blood cells by red pulp macrophages was dependent on the same Fc?Rs as phagocytosis by blood monocyte-derived macrophages, especially in regarding the response to IV immunoglobulin. Concluding, we show the distinct nature of splenic red pulp macrophages in human subjects. Knowledge on the Fc?R expression and usage of these cells is important for understanding and improving treatment strategies for autoimmune diseases such as ITP and AIHA.
Project description:Tissue macrophages comprise a heterogeneous group of cell types differing in location, surface markers and function. Red pulp macrophages are a distinct splenic subset involved in removing senescent red blood cells. Transcription factors such as PU.1 (also known as Sfpi1) and C/EBPalpha (Cebpa) have general roles in myelomonocytic development, but the transcriptional basis for producing tissue macrophage subsets remains unknown. Here we show that Spi-C (encoded by Spic), a PU.1-related transcription factor, selectively controls the development of red pulp macrophages. Spi-C is highly expressed in red pulp macrophages, but not monocytes, dendritic cells or other tissue macrophages. Spic(-/-) mice have a cell-autonomous defect in the development of red pulp macrophages that is corrected by retroviral Spi-C expression in bone marrow cells, but have normal monocyte and other macrophage subsets. Red pulp macrophages highly express genes involved in capturing circulating haemoglobin and in iron regulation. Spic(-/-) mice show normal trapping of red blood cells in the spleen, but fail to phagocytose these red blood cells efficiently, and develop an iron overload localized selectively to splenic red pulp. Thus, Spi-C controls development of red pulp macrophages required for red blood cell recycling and iron homeostasis.
Project description:A main function of splenic red pulp macrophages is the degradation of damaged or aged erythrocytes. Here we show that these macrophages accumulate ferrimagnetic iron oxides that render them intrinsically superparamagnetic. Consequently, these cells routinely contaminate splenic cell isolates obtained with the use of MCS, a technique that has been widely used in immunological research for decades. These contaminations can profoundly alter experimental results. In mice deficient for the transcription factor SpiC, which lack red pulp macrophages, liver Kupffer cells take over the task of erythrocyte degradation and become superparamagnetic. We describe a simple additional magnetic separation step that avoids this problem and substantially improves purity of magnetic cell isolates from the spleen.
Project description:To compare the splenic macrophages between SIRPα-knockout mice and WT mice, we performed a complete transcript profiling of the splenic red pulp macrophages from SIRPα-KO mice compared to WT mice using mRNA microarray as a discovery platform. SIRPα-KO mice and WT mice were kept under the same condition. Macrophages were isolated from spleen red pulp of SIRPα-KO mice and WT mice. RNA was then isolated from the same number of freshly isolated macrophages. Overall design: In each group, 5 mice were used. Splenic red pulp macrophages isolated from 5 SIRPα-KO or 5 WT mice were mixed before RNA extraction.
Project description:The cholinergic anti-inflammatory pathway (CAP) links the nervous and immune systems and modulates innate and adaptive immunity. Activation of the CAP by vagus nerve stimulation exerts protective effects in a wide variety of clinical disorders including rheumatoid arthritis and Crohn's disease, and in murine models of acute kidney injury including ischemia/reperfusion injury (IRI). The canonical CAP pathway involves activation of splenic alpha7-nicotinic acetylcholine receptor (?7nAChR)-positive macrophages by splenic ?2-adrenergic receptor-positive CD4+ T cells. Here we demonstrate that ultrasound or vagus nerve stimulation also activated ?7nAChR-positive peritoneal macrophages, and that adoptive transfer of these activated peritoneal macrophages reduced IRI in recipient mice. The protective effect required ?7nAChR, and did not occur in splenectomized mice or in mice lacking T and B cells, suggesting a bidirectional interaction between ?7nAChR-positive peritoneal macrophages and other immune cells including ?2-adrenergic receptor-positive CD4+ T cells. We also found that expression of hairy and enhancer of split-1 (Hes1), a basic helix-loop-helix DNA-binding protein, is induced in peritoneal macrophages by ultrasound or vagus nerve stimulation. Adoptive transfer of Hes1-overexpressing peritoneal macrophages reduced kidney IRI. Our data suggest that Hes1 is downstream of ?7nAChR and is important to fully activate the CAP. Taken together, these results suggest that peritoneal macrophages play a previously unrecognized role in mediating the protective effect of CAP activation in kidney injury, and that Hes1 is a new candidate pharmacological target to activate the CAP.
Project description:Splenic myelopoiesis provides a steady flow of leukocytes to inflamed tissues, and leukocytosis correlates with cardiovascular mortality. Yet regulation of hematopoietic stem cell (HSC) activity in the spleen is incompletely understood. Here, we show that red pulp vascular cell adhesion molecule 1 (VCAM-1)(+) macrophages are essential to extramedullary myelopoiesis because these macrophages use the adhesion molecule VCAM-1 to retain HSCs in the spleen. Nanoparticle-enabled in vivo RNAi silencing of the receptor for macrophage colony stimulation factor (M-CSFR) blocked splenic macrophage maturation, reduced splenic VCAM-1 expression and compromised splenic HSC retention. Both, depleting macrophages in CD169 iDTR mice or silencing VCAM-1 in macrophages released HSCs from the spleen. When we silenced either VCAM-1 or M-CSFR in mice with myocardial infarction or in ApoE(-/-) mice with atherosclerosis, nanoparticle-enabled in vivo RNAi mitigated blood leukocytosis, limited inflammation in the ischemic heart, and reduced myeloid cell numbers in atherosclerotic plaques.
Project description:Tissue-resident macrophages in the spleen, including red pulp and white pulp macrophages, marginal zone macrophages (MZMs) and marginal zone metallophilic macrophages (MMMs), are highly heterogeneous as a consequence of adaptation to tissue-specific environments. Each macrophage sub-population in the spleen is usually identified based on the localization, morphology and membrane antigen expression by immunohistochemistry. However, their phenotypical and functional characteristics remain incompletely understood due to the difficulty of identification and isolation by flow cytometry. We used a cocktail of three enzymes (Collagenase D, Dispase I and DNase I), rather than traditional mechanical grinding, for isolation of each sub-population, which resulted in significant improvement of isolation of these macrophage sub-populations, particularly MZMs and MMMs, as determined by CD11bhiF4/80medTim4hi and CD11bhiF4/80medTim4med, respectively. This method should be helpful for molecular and functional characterization of each splenic resident macrophage sub-population.
Project description:The spleen plays an integral protective role against encapsulated bacterial infections. Our understanding of the associated mechanisms is limited to thymus-independent (TI) antibody production by the marginal zone (MZ) B cells, leaving the contribution of other splenic compartments such as the red pulp (RP) largely unexplored despite asplenic patients succumbing to the infection in the first 24 h, suggesting important antibody-independent mechanisms. In this study, using time-lapse intravital imaging of the spleen, we identify a tropism for Streptococcus pneumoniae in this organ mediated by tissue-resident MZ and RP macrophages and a protective role for two distinct splenic neutrophil populations (Ly6Ghi and Ly6Gintermediate) residing in the splenic RP. Splenic mature neutrophils mediated pneumococcal clearance in the spleen by plucking bacteria off the surface of RP macrophages that caught the majority of bacteria in a complement-dependent manner. This neutrophil phagocytic capacity was further enhanced after TI antibody production. Resident immature neutrophils (Ly6Gintermediate) in the spleen undergo emergency proliferation and mobilization from their splenic niche after pneumococcal stimulation to increase the effector mature neutrophil pool. We demonstrate that splenic neutrophils together with two macrophage populations and MZ B cells regulate systemic S. pneumoniae clearance through complementary mechanisms.
Project description:Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) - frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) - were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2, TNFAIP3 and MYD88, common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent (TNFAIP3 and MYD88) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia.