Lactation-associated macrophages exist in murine mammary tissue and human milk

Macrophages are involved in immune defense, organogenesis and tissue homeostasis. Macrophages contribute to the different phases of mammary gland remodeling during development, pregnancy and involution postlactation. Less is known about the dynamics of mammary gland macrophages in the lactation stage. Here, we describe a macrophage population present during lactation in mice. By multiparameter flow cytometry and single-cell RNA sequencing, we identified a lactation-induced CD11c+CX3CR1+Dectin-1+ macrophage population (liMac) that was distinct from the two resident F4/80hi and F4/80lo macrophage subsets present pregestationally. LiMacs were predominantly monocyte-derived and expanded by proliferation in situ concomitant with nursing. LiMacs developed independently of IL-34, but required CSF-1 signaling and were partly microbiota-dependent. Locally, they resided adjacent to the basal cells of the alveoli and extravasated into the milk. We found several macrophage subsets in human milk that resembled liMacs. Collectively, these findings reveal the emergence of unique macrophages in the mammary gland and milk during lactation.

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Software and code
Policy information about availability of computer code Data collection Flow cytometry data were collected using FACS Diva Software v9.1 or SpectroFlo® Software version 3.1.0.
Microscopy images were acquired using LAS X software.

Data analysis
FlowJo 10.6.2 and 10.8.1 (Tree star) was used for the Flow cytometry data analysis; R and R studio v3.2-v4.0 was used for bioinformatics analysis; UMAP and FlowSOM algorithms were used from publicly available sources, which are cited in the paper; GraphPad Prism v 7.03 and 9.4.0 (Graphpad Software Inc.) was used to prepare graphs and perform statistical analysis; LAS X (Leica) and Imaris (Bitplane) software were used for image analysis; Seurat v4.1 in conjunction with Platypus v3.4.1 for scRNAseq general analysis, plots and Tabula Muris data preparation and annotation transfer; Symphony v0.1.0 for integration with Tabula Muris; Limma, edgeR and 3dVolcano for polar volcano. The analysis code has been uploaded to https://github.com/gustaveroussy/FG-Lab.
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April 2020
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Publicly available datasets: GRCm38.p6 Release M23, GRCh38.p13, Ensembl database, MoMAC-VERSE. The mouse mammary gland and human milk scRNA-seq datasets are deposited in the Genome Expression Omnibus under the accession numbers: GSE230697 and GSE230749, respectively. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD041711.

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Sample size
The sample size was chosen according to 3R principles, and normally included 3-5 mice per group, which is usually enough to get a statistical difference should there be any. No statistical method to predetermine sample size was used. Details on sample sizes are provided in figure legends and methods section.
Data exclusions No animals were excluded from analysis unless re-genotyping revealed a wrongly determined genotype: then the animal was re-allocated in a correct group. FACS data were pregated for certain markers as described in figure legends. For scRNAseq data analysis, low quality cells exclusion and doublets filtering was done as described in methods section.

Replication
All experiments were independently repeated at least once and the number of experiments was stated in the figure legends. Single cell RNA sequencing analysis was performed once.
Randomization No special method of randomization was used. The mice were randomly allocated to cages upon arrival from vendour or weaning. Covariates influence was controlled by keeping mice in the same conditions and repeating experiments several times. Experimental and control groups were distributed to equal numbers.

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It was not possible to make the investigators completely blinded to group allocation, since usually the same experimenter performed genotyping, experimental procedures, tissue harvest and data analysis, however, whenever possible, experimenters tried not to pay attention to experimental group identity before the final steps of data analysis.

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No wild animals were used in the study.
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All experimental procedures at the University of Zurich were performed in accordance with Swiss Federal regulations and approved by the Cantonal Veterinary Office of Zurich.
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Population characteristics
Human milk samples were collected at the University Hospital Zurich from healthy women, aged 20 to 40 years [mean (SD) 32.0 (5.0)], within 4 to 63 days after delivery.

Recruitment
The recruitment of the study participants and the collection of human milk samples took place at the Department of Neonatology of the University Hospital Zurich. Women eligible as study participants were contacted by the project leader, informed about the present study protocol and asked for consent for participating to the study. The informed consent process included ample time for consideration given to the participants and opportunity to ask questions. No compensation or payments were given to the study participants. There were no selection biases. The milk donors could withdraw their consent at any point in time without justification.

Ethics oversight
The Federal Act on Research involving Human Beings (HRA, RS 810.30) and the Ordinance on Human research with the exception of clinical trials (HRO,RS 810.301,. The study protocol was approved by the Swiss Ethics Commission of the Canton of Zurich (BASEC-Nr. 2020-00542) and all the subjects participating to the study signed an inform consent before the enrollment.
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nature research | reporting summary
April 2020 Flow Cytometry Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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Methodology
Sample preparation Mice were sacrificed by CO2 inhalation and intracardially perfused with phosphate buffered saline (PBS; pH 7.4, Gibco). After perfusion, left and right abdominal mammary glands were dissected, and inguinal lymph nodes removed. All the samples were cut into small pieces in an Eppendorf tube, followed by digestion in 0.4 mg/ml collagenase type IV (Worthington) and 0.04 mg/ml DNase I in Ca2+/Mg2+ HBSS supplemented with 10% FCS for 40 mins at 37°C, while shaking. Samples were homogenized with a 18 G needle and a syringe and filtered through a 100 μm cell strainer to obtain a homogeneous cell suspension. Cells were once washed in PBS, resuspended in red blood cell lysis (0.16 M NH4Cl, 0.11 M KHCO3, 0.001 M EDTA in ddH2O) and incubated on ice for 5 min, then filtered through 100 μm cell strainer and washed with PBS.
For isolation of milk cells, milk was diluted in a 1:1 ratio with PBS and centrifuged at 800 g for 20 minutes at 15°C. The lipid layer and skim milk were removed, and the cell pellet was washed twice in PBS by centrifugation at 400 g for 5 minutes and resuspension in PBS.
For histology, mice were euthanized through CO2 asphyxiation and perfused with PBS. Mammary fat pads were removed, fixed in 4% PFA (Morphisto) for 6-24 hours at 4°C, washed in PBS followed by incubation in 30% sucrose in PBS at 4°C for 1 to 5 days. The tissue was then embedded in Cryo Embedding Medium (Medite) and frozen on dry ice.

Instrument
Flow cytometry analysis was performed on LSR II Fortessa, BD FACSymphony and Cytek Aurora. Samples for scRNAseq were sorted on Aria III and S6 cell sorters. Sequencing was performed on NovaSeq6000 platform. Imaging was performed on Leica SP8 Falcon and Leica SP5 microscopes.

Gating strategy
In general, cells were gated based on FSC-A and SSC-A to exclude debris, doublets were excluded by FSC-Area vs. FSC-Height gating. Dead cells were excluded from the analysis using Zombie NIR fixable staining reagent (BioLegend). Supplemental gating strategies are provided in Extended Data.
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