MafB-restricted local monocyte proliferation precedes lung interstitial macrophage differentiation

Resident tissue macrophages (RTMs) are differentiated immune cells that populate distinct niches and exert important tissue-supportive functions. RTM maintenance is thought to rely either on differentiation from monocytes or on RTM self-renewal. Here, we used a mouse model of inducible lung interstitial macrophage (IM) niche depletion and refilling to investigate the development of IMs in vivo. Using time-course single-cell RNA-sequencing analyses, bone marrow chimeras and gene targeting, we found that engrafted Ly6C+ classical monocytes proliferated locally in a Csf1 receptor-dependent manner before differentiating into IMs. The transition from monocyte proliferation toward IM subset specification was controlled by the transcription factor MafB, while c-Maf specifically regulated the identity of the CD206+ IM subset. Our data provide evidence that, in the mononuclear phagocyte system, the ability to proliferate is not merely restricted to myeloid progenitor cells and mature RTMs but is also a tightly regulated capability of monocytes developing into RTMs in vivo.

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection Bulk RNA-seq: Sequence alignment with the mouse genome (GRCm38), sequence counting and quality control were performed using the nfcore/rnaseq pipeline. scRNA-seq: Cell Ranger (v3.0.2) (10x Genomics) was then used to demultiplex the BCL files into FASTQ files (cellranger mkfastq), to perform alignment (to Cell Ranger human genome references 3.0.2 GRCm38/build 97), filtering, UMI counting and to produce gene barcode matrices (cellranger count). For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

March 2021
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 description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Single-cell RNA-seq and bulk RNA-seq data have been deposited at GEO and are publicly available under GEO accession GSE 194021. All original codes have been deposited at GitHub and are available via this link: https://github.com/BlanQwall/Lung_IM_differentiation. Any additional information required to reanalyze the data reported in this paper is available from the corresponding author upon request.

Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.
Reporting on sex and gender Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
No statistical methods were used to pre-determine sample sizes but our sample sizes are similar to those reported in previous publications (PMID: 28329706; PMID: 31481690; PMID: 31591573). Data distribution was assumed to be normal when parametric tests were performed, but this was not formally tested.
Data exclusions No data were excluded from the analyses.

Replication
For each experiment, each experimental group was composed of 3-5 mice constituting biological replicates. Each experiments have been repeated two to four times. All attempts at replication were successful and gave similar readout.
Randomization Mice were identified according to genotype and all experiments were performed with age-and sex-matched littermates. For Csf1r blocking experiments mice were randomly assigned to vehicle or isotype Ab and anti-Csf1r treatments. For experiments using IMDTR mice that were treated or not with DT, mice were randomly allocated to DT treatment or not.

Blinding
Investigators were not blinded during the collection and analysis of the data, except for the quantification of microscopy lung sections, where investigators were blinded.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. TotalSeq™-A0306 anti-mouse Hashtag 6 Antibody;BioLegend;155811 TotalSeq™-A0307 anti-mouse Hashtag 7 Antibody;BioLegend;155813 TotalSeq™-A0308 anti-mouse Hashtag 8 Antibody;BioLegend;155815 All antibodies were used at 1:100 dilution as recommended by BioLegend.

Validation
All the antibodies are from commercial sources that perform rigorous testing for specificity, quality control and lot to lot variability. Specific statements of validation include: BD bioscience https://www.bdbiosciences.com/en-us/products/reagents/flow-cytometry-reagents/research-reagents/quality-and-reproducibility The specificity is confirmed using multiple methodologies that may include a combination of flow cytometry, immunofluorescence, immunohistochemistry or western blot to test staining on a combination of primary cells, cell lines or transfectant models. The manufacturing process for the BD Biosciences antibodies and reagents adheres to standard operating procedures (SOPs) and guidelines to ensure lot-to-lot consistency. All flow cytometry reagents are titrated on the relevant positive or negative cells.
BioLegend https://www.biolegend.com/en-us/quality/quality-control Flow cytometry reagents: Specificity testing of 1-3 target cell types with either single-or multi-color analysis (including positive and negative cell types). Once specificity is confirmed, each new lot must perform with similar intensity to the in-date reference lot. Brightness (MFI) is evaluated from both positive and negative populations. Each lot product is validated by QC testing with a series of titration dilutions. TotalSeq Antibodies: Bulk lots are tested by PCR and sequencing to confirm the oligonucleotide barcodes. They are also tested by flow cytometry to ensure the antibodies recognize the proper cell populations. Bottled lots are tested by PCR and sequencing to confirm the oligonucleotide barcodes.

Animals and other research organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research, and Sex and Gender in Research

March 2021
Cx3cr1LSL-DTR/+ (The Jackson Laboratory, Strain #025629), Ccr2-/-(The Jackson Laboratory, Strain#004999), Nr4a1-/-(The Jackson Laboratory, Strain #006187), Maffl/fl (kindly provided by Dr. Fabienne Andris), Mafbfl/fl (generated in-house, see methods), Lyz2Cre (The Jackson Laboratory, Strain #004781) and Ms4a3Cre (kindly provided by Dr. Florent Ginhoux). Myeloid-restriced Maf or Mafb depletion was achieved by crossing Maffl/fl or Mafbfl/fl mice with Lyz2Cre or Ms4a3Cre mice. A mix of male and female mice between 6 and 10 weeks of age were used for each experiment, except for chimera experiments where mice between 11 and 15 weeks of age were used. The mice were bred and housed under specific pathogen-free conditions at the GIGA Institute (Liège University, Belgium), maintained in a 12-h light-dark cycle, and had access to normal diet chow and water ad libitum. Mice were identified according to genotype and all experiments were performed with age-and sex-matched littermates. For Csf1r blocking experiments mice were randomly assigned to vehicle or isotype Ab and anti-Csf1r treatments. For experiments using IMDTR mice that were treated or not with DT, mice were randomly allocated to DT treatment or not.

Wild animals
The study did not involve wild animals.

Reporting on sex
No sex-specific differences were observed in pilot experiments. A mix of male and female mice between 6 and 10 weeks of age were used for each experiment, except for chimera experiments where mice between 11 and 15 weeks of age were used.
Field-collected samples The study did not involve samples collected from the field.

Ethics oversight
All animal experiments described in this study were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Liège (Ethical Approval #DE1956). The 'Guide for the Care and Use of Laboratory Animals,' prepared by the Institute of Laboratory Animal Resources, National Research Council, and published by the National Academy Press, as well as European and local legislations, were followed carefully. Accordingly, the temperature and relative humidity were 21°C and 45-60%, respectively.
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Blood was collected by retro-orbital plexus bleeding of terminally-anesthetized mice. Mice were then euthanized by cervical dislocation. Peritoneal lavage was obtained by injecting 10 mL HBSS (Lonza, Cat#BE10-508F) into the peritoneal cavity and collecting the washout. Mice were then perfused with 10 mL PBS via the left ventricle and lungs, brain, liver, spleen, intestine and colon were dissected. For BM cells, femurs and tibias were dissected and cleaned of soft adhering tissue. Distal and proximal ends were opened, and BM cells were flushed out. After centrifugation, cell pellets were re-suspended in ice-cold PBS (ThermoFisher, Cat#14190094) containing 10 mM of EDTA (Merck Millipore, Cat#1084181000) and cell suspensions were filtered using a cell strainer (70 μM, Corning, Cat#352350) to obtain a single cell suspension. Lungs, brains, liver and spleen were cut into small pieces with razor blades, and digested for 1 h at 37 °C in HBSS containing 5% v/v of FBS (ThermoFisher, Cat#10270098), 1 mg/mL collagenase A (Sigma, Cat#14190094) and 0.05 mg/mL DNase I (Sigma, Cat#11284932001). After 45 min of digestion, the suspension was flushed using a 18 G needle to dissociate aggregates. Ice-cold PBS (ThermoFisher, Cat#) containing 10 mM of EDTA (Merck Millipore, Cat#1084181000) was added to stop the digestion process and cell suspensions were filtered using a cell strainer (70 μM, Corning, Cat#352350). Mononuclear leukocytes from lungs and livers were enriched using a Percoll density gradient (GE Healthcare, Cat#17089101) and harvesting cells from the 1.080:1.038 g/mL interface. For the isolation of leukocytes from the small intestines and colons, small intestines and colons were dissected from the pylorus and the rectum, were separated from the mesenteric tissue from Peyer's patches and from fat and were placed in ice-cold HBSS with 2% FBS. Intestinal content was removed with PBS, and the small intestines and colons were opened by a longitudinal cut and washed 3 times in ice-cold HBSS with 2% FBS. To remove mucus and epithelial cells, small intestines and colons were incubated with HBSS with 2% FBS and 1 mM 1,4 dithiothreitol (DTT, Sigma, 10197777001) for 20 min with constant shaking followed by an incubation with HBSS containing 2% FBS and 1.3mM EDTA for 40 min. Tissue pieces were then cut into small pieces and incubated for 1 h at 37 C with RPMI containing 2% FBS, 2 mg/mL collagenase IV (ThermoFisher, Cat#17104019) and 40 U/mL DNase I. At the end of incubation, the suspension was homogenized with a 19G syringe and filtered through a 70 μM strainer.

Instrument
Cell suspensions were analyzed with a LSRFortessa (BD Biosciences). For scRNA-seq and bulk RNA-seq, lung myeloid cells were sorted using an FACSAriaIII (BD Biosciences).

Software
Results were analyzed using FlowJo software (Tree Star Inc.).

Cell population abundance
Purity was between 90 and 95% and was determined by flow cytometry after sorting.