immunology.sciencemag.org/cgi/content/full/4/35/eaau3814/DC1

Supplementary Materials for

RELMα-expressing macrophages protect against fatal lung damage

and reduce parasite burden during helminth infection

Branislav Krljanac, Christoph Schubart, Ronald Naumann, Stefan Wirtz, Stephan Culemann, Gerhard Krönke, David Voehringer

*Corresponding author. Email: david.voehringer@uk-erlangen.de

Published 24 May 2019, Sci. Immunol. 4, eaau3814 (2018)

DOI: 10.1126/sciimmunol.aau3814

The PDF file includes:

Raw data Methods Fig. S1. Generation of RetnlaCre mice. Fig. S2. Structure and sequence information on the recombined BAC vector and its copy number in the genome. Fig. S3. RELMα_tdTomato expression correlates with Retnla mRNA and RELMα protein expression and can be induced in vitro by IL-4. Fig. S4. Immunofluorescence analysis of RELMα_tdTomato expression in selected organs of RetnlaCre_R26tdTomato mice. Fig. S5. Ex vivo RELMα_tdTomato fluorescence imaging of selected organs of naïve RetnlaCre_R26tdTomato mice. Fig. S6. Flow cytometry gating strategies for the indicated cell types isolated from naïve RetnlaCre_R26tdTomato mice. Fig. S7. Analysis of RELMα_tdTomato-expressing Alv-MΦs in RetnlaCre_R26tdTomato mice. Fig. S8. RELMα_tdTomato expression in MΦs derived from adoptively transferred monocytes. Fig. S9. Analysis of MΦs in the lungs of RetnlaCre_R26tdTomato mice infected with N. brasiliensis. Fig. S10. Depletion efficiency of RELMα_tdTomato+ cells after DT treatment of RetnlaCre_R26tdTomato/iDTR mice. Table S1. List of antibodies used for flow cytometry and immunofluorescence. Table S2. Sequences of primers used for quantitative RT-PCR. References (42–45)

Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/4/35/eaau3814/DC1)

Raw data (Microsoft Excel format)

Supplemental Material

Supplemental Methods

Generation of RetnlaCre mice

To generate RetnlaCre bacterial artificial chromosome (BAC)-transgenic mice, BAC clone

RP23-463N16 (RPCI-23 C57BL/6J Mouse BAC Library; BACPAC Resource Center at

Children's Hospital Oakland Research Institute) was first modified by sequentially removing

Retnlg and Retnlb genes (flanking the Retnla gene) via homologous recombination-based

genetic engineering and galK-mediated selection (galK expression plasmid obtained as gift

from NCI Frederick) in SW105 E. coli strain, essentially as described (42). To insert gene

encoding Cre recombinase behind the start codon of the Retnla gene, the BAC clone was

further modified by a targeting vector containing Cre-Frt-Neo-Frt cassette flanked by two

short (300-400 bp) homologous sequences. Homologous recombination was induced by a

temperature shift and bacteria were grown in arabinose-containing medium to induce flippase-

mediated excision of the Neo-cassette (Fig. S1). Lastly, the BAC clone was linearized,

purified on a gel filtration column (Sepharose CL-4B; Sigma-Aldrich) in microinjection

buffer (43) and injected into pronuclei of fertilized C57BL/6 oocytes. For generating targeting

constructs, GeneArt seamless plus cloning and assembly kit (Invitrogen) was used and correct

BAC modifications as well as the purity of the final construct were verified by restriction

enzyme digestion and pulsed-field gel electrophoresis.

Generation of bone marrow (BM) chimeras

BM cells were prepared from femur and tibia of donor mice and resuspended in RPMI-1640

(GibcoTM at Thermo Fisher Scientific, Waltham, MA). Then, 3x106 mixed bone marrow cells

from C57BL/6 Ly5.1 mice were transferred i.v. into C57BL/6 Ly5.2 RetnlaCre_R26tdTomato

recipient mice (and vice versa) that had been lethally irradiated with one dose of 600 rad and

one dose of 500 rad given 4 h apart. Mice were provided antibiotics (2 g/L neomycin sulfate,

100 mg/L polymyxin B sulfate; Sigma-Aldrich, St. Louis, MI) in drinking water for 8 weeks

after the bone marrow transfer.

Monocyte transfers

Monocytes were sorted from BM of respective donor mice using the EasySep mouse

monocyte isolation kit (Stemcell Technologies, Vancouver, BC) and labeled with CellTrace

Violet (Molecular ProbesTM at Thermo Fisher Scientific) according to the manufacturer’s

instructions. Monocytes were injected in 200 µl RPMI-1640 typically at 0,5x106

cells/recipient (for i.p. transfer) and 1,5x106 cells/recipient (for i.v. transfer on day 3 post N.

brasiliensis infection). Analysis of recipient mice was performed on day 6 after i.p or day 37

post i.v. transfer.

Diphtheria toxin (DT) injections

DT was purchased from Sigma-Aldrich and injected into mice i.p. at a dose of 25 ng/gram of

body weight (~600 ng per mouse) in 200 µL RPMI-1640. To estimate cell depletion, mice

were injected with DT at 0 and 24 h and blood and peritoneal exudates were harvested on day

2 p.i. For depleting MΦs during N. brasiliensis infections, mice were injected with DT on

days 0, 1, 2 and 3 or 4, 5, and 7 after primary or at days 0, 1, 2, 3 and 4 after secondary

infection.

In vitro cell culture and stimulation of macrophages

For harvesting peritoneal and alveolar macrophages, lavage was performed with 10 ml or 3 x

1 ml cold PBS, respectively. For generating BMDMs, BM cells were resuspended in DMEM

supplemented with 10% fetal calf serum, 2mM L-glutamine, 100 U/mL penicillin, and 100

µg/mL streptomycin (all from GibcoTM). After removing adherent cells in cell culture treated

flasks for 2h at 37°C, nonadherent bone marrow cells were cultivated in non-treated petri

dishes (Sarstedt, Nümbrecht, Germany) or ultra-low attachment 24-well plates (Corning,

Corning, NY) for 7 days in the presence of 20% supernatant from the macrophage colony-

stimulating factor producing fibroblast L929 cell line. Macrophages (>90% purity) were

detached from Petri dishes using Accutase (Innovative Cell Technologies, San Diego, CA)

according to the manufacturer’s instructions, washed, counted, and replated at a density of 106

cells/mL for 24-72 hours with medium (control), IL-4 (20 ng/mL; R&D Systems,

Minneapolis, MN), or lipopolysaccharide (100 ng/mL; Sigma-Aldrich) and interferon gamma

(10 ng/mL; PeproTech, London, UK).

Flow cytometry

Tissues were digested with liberase (100 µg/mL; Roche Diagnostics, Rotkreuz, Switzerland)

in PBS for 45 min at 37°C and mechanically disrupted in a 70 µm nylon strainer (Becton

Dickinson, Franklin Lakes, NJ). For white adipose tissue, liver, and ileum samples,

leukocytes were enriched by mixing equal ratio of cells and 1,07 g/mL stock isotonic percoll

solution (GE Healthcare Life Sciences, Pittsburgh, PA). Brain samples were processed with

neural tissue dissociation kit (P) and myelin removal beads II (Miltenyi Biotec, Bergisch

Gladbach, Germany). Thymus samples were stained with biotinylated anti-CD3 and

processed with EasySep mouse biotin positive selection kit (Stemcell Technologies) to

remove T cells. Single-cell suspensions were incubated with CD16/32 blocking antibody

(clone 2.4G2; BioXcell, West Lebanon, NH) followed by staining with diluted antibody

mixtures at 4°C. A list of primary and secondary antibodies used is given in the

supplementary Table S1. For intracellular staining cells were fixed in 4% paraformaldehyde

in PBS and permeabilized with IC fixation and permeabilisation buffer or for dual RELMα

and tdTomato visualization in 1% paraformaldehyde in PBS and permeabilized using the

Foxp3/Transcription factor staining buffer set (all from eBioscience at Thermo Fisher

Scientific). For measuring proliferation, cells were labeled with CellTrace Violet (Molecular

ProbesTM) according to the manufacturer’s instructions. Samples were acquired on FACS

Canto II (BD Biosciences, San Jose, CA) and analyzed with FlowJo software (TreeStar,

Ashland, OR).

Immunofluorescence

Cryosections (8 µm thick) of paraformaldehyde-fixed tissue samples were blocked with 2,5%

bovine serum albumin in PBS supplemented with 5% normal donkey serum. Whole mount

tissue samples were prepared essentially as described previously (44, 45). Cryosections were

stained with antibodies in blocking buffer, with the addition of permeabilisation buffer

(eBioscience) for whole mount samples. A list of primary and secondary antibodies used is

given in the supplementary Table S1. Adipocytes were stained with BODIPY 493/503 dye (2

µg/mL; Molecular ProbesTM) and nuclei were counterstained with 4′,6-Diamidino-2-

phenylindole dihydrochloride (DAPI). Pictures were acquired on LSM700 Observer.Z1 or

Axio Vert.A1 and image analysis was performed using the ZEN2 Ver.10.0 imaging software

(all Carl Zeiss, Jena, Germany).

Quantitative RT-PCR

Macrophages were sorted from bronchoalveolar (FSChi CD11chi) or peritoneal lavage fluid

(FSChi CD11bhi), and neutrophils (Ly6G+ Ly6C+) from blood of naïve RetnlaCre_R26tdTomato

mice. Int-MΦs/DCs from N. brasiliensis-infected lungs were positively selected using

biotinylated anti-CD11c (N418) and EasySep biotin positive selection kit (Stemcell

Technologies), followed by sorting for MHC-II+ cells. All cells were finally gated as

tdTomato+ or tdTomato− populations and sorted using S3e Cell Sorter (Bio-Rad, Hercules,

CA). RNA was isolated from sorted cells or tissues with RNeasy Mini or Micro Kit (Qiagen,

Hilden, Germany), respectively. For skin probes, tissue was grinded with mortar and pestle

chilled with liquid nitrogen, and RNA was isolated with TRIsure reagent (Bioline, London,

UK). Then, 1 µg total RNA was used for first-strand cDNA synthesis using MultiScribe

Reverse Transcriptase and random primers (Applied Biosystems, Foster City, CA). PCR with

complementary DNA templates was performed using the SYBR Select Master Mix (Applied

Biosystems) or SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and the CFX

Connect Real-Time System (Bio-Rad). Sequences of primers used for quantitative RT-PCR

are given in the supplementary Table S2. Pbgd or Hprt were used as reference genes and PCR

conditions were the following: 10 min initial denaturation at 95°C, 40 cycles with 30 s of

denaturation at 95°C, 30 s of annealing at 60°C, and 45 s of elongation at 72°C.

Supplemental Figures

Fig. S1. Generation of RetnlaCre mice. (A) Localization of genes present within the bacterial artificial chromosome (BAC) clone RP23-463N16 used to generate RetnlaCre mice (upper scheme). In the final targeting construct, coding sequences of Retnlg and Retnlb genes which are flanking the Retnla gene of interest are deleted, whereas the gene encoding Cre recombinase is placed directly behind the start codon of Retnla (bottom scheme). A truncated Trat1 gene is present upstream of Retnlb on this BAC clone. (B) To remove Retnlg (example shown) and subsequently Retnlb gene from the original BAC clone, the respective gene was replaced with a galK expression cassette (see experimental procedures) which was then also eliminated using the same pair of short (300-400 bp) flanking homologous sequences. (C) To insert the coding sequence of the Cre recombinase upstream of the start codon (ATG) of Retnla, a targeting vector containing a Cre-Frt-Neo-Frt cassette flanked by two short homologous sequences was used. We electroporated this targeting vector into the SW105 E. coli strain containing the modified BAC with deleted Retnlg and Retnlb genes. After homologous recombination, bacteria were grown in arabinose-containing medium to induce flippase-mediated excision of the Neo-cassette.

Fig. S2. Structure and sequence information on the recombined BAC vector and its copy number in the genome. (A) Location of the Cre-cassette (pink) within exon 1 of Retnla gene after deletion of the FRT-flanked Neo resistance gene. Retnla exons are shown in red and numbers indicate the basepairs. (B) Sequence information for the start of the integrated Cre cassette. (C) location of in-frame STOP codons downstream of the Cre cassette. (D and E) Location of the 5’- and 3’-homology arms used to delete the Retnlb (D) and Retnlg (E) genes. (F) qPCR results for determination of the BAC copy number in the genome of Retnla-Cre mice. Retnla exon 4 was amplified with perimers (Retnla-Exon4-fw: 5‘-GGATGACTGCTACTGGGTGTGC-3‘ and -rev: 5‘-CAAGAAGCATTTCAAGAAGCAGGG-3‘) and its C(t) values were normalized to C(t) values for the STAT6 gene amplified with primers (STAT6-WT-fw: 5´-ACTCGGAAAGCCTCATCTT-3´ and -rev: 5´-AAGTGGGTCCCCTTCACTCT-3´). The bars show the mean+SD of 3 mice per group and indicate that the BAC vector integrated with 5 copies into the genome.

F

Fig. S3. RELMα_tdTomato expression correlates with Retnla mRNA and RELMα protein expression and can be induced in vitro by IL-4. (A) Expression of Retnla mRNA (normalized to Pbgd mRNA) in sorted RELMα_tdTomato+ (black bars) and RELMα_tdTomato– (white bars) peritoneal MΦs (pΜΦs) and blood neutrophils of naïve RetnlaCre_R26tdTomato mice, analyzed by quantitative RT-PCR. (B) Correlation of RELMα and RELMα_tdTomato expression in small pMΦs (SPM) and large pMΦs (LPM) of naïve mice. (C) Total lung cells of naïve RetnlaCre_R26tdTomato mice were pre-gated as CD68+ MHC-II+, further sub-gated into CD11bhigh CD11+ interstitial MΦs (Int-MΦs) and CD11blow CD11chigh lung DCs and analyzed for RELMα_tdTomato expression. The histogram panels show percentages of Int-MΦs and lung DCs expressing intracellular RELMα protein within RELMα_tdTomato+ (red histogram) and RELMα_tdTomato– populations (blue histogram), respectively. (D) Bone marrow-derived MΦs (BMDMs) were generated from RetnlaCre_R26tdTomato mice and stimulated with 20 ng/ml of recombinant murine IL-4 in vitro for the indicated time points. Numbers indicate the percentages of BMDMs expressing intracellular RELMα protein. (E) BMDMs derived from 5 independent RetnlaCre_R26tdTomato founder lines were stimulated with IL-4 as in (D) and RELMα_tdTomato expression was analyzed by flow cytometry at the indicated time points post-stimulation. Compared to data in (D), founder lines 7 and 13 exhibited similar RELMα_tdTomato to endogenous RELMα protein expression kinetics. (F) BMDMs of WT and STAT6-deficient RetnlaCre_R26tdTomato mice (STAT6ko) were either left untreated (UN) or stimulated with IL-4 or lipopolysaccharide (LPS) plus interferon gamma (IFNg) in vitro and RELMα_tdTomato expression was analyzed by flow cytometry 48h post-stimulation.

Fig. S4. Immunofluorescence analysis of RELMα_tdTomato expression in selected organs of RetnlaCre_R26tdTomato mice. (A) RELMα_tdTomato indicates endogenous RetnlaCre-induced expression of the tdTomato fluorescent reporter from the Rosa26 locus (red), anti-CD68 staining was used to label tissue MΦs (green), and DAPI for nuclei (blue). Images on the far right show overlay of the respective channels (merge). Scale bars = 50 µm. (B) Immunofluorescence analysis of the lung of naïve (upper) and day 7 Nb-infected (lower) RetnlaCre_R26tdTomato mice. For comparison, isotype control stainings for the anti-RELMα antibody are also shown. Scale bar = 50 µm.

Fig. S5. Ex vivo RELMα_tdTomato fluorescence imaging of selected organs of naïve RetnlaCre_R26tdTomato mice. (A) Selected organs of RetnlaCre –_R26tdTomato (left organ) and RetnlaCre+_R26tdTomato mice (right organ) were imaged under visible (black/white images) or green light (tdTomato fluorescence analyzed at 620nm) using the Maestro in vivo imaging system (Intas Science Imaging). Signals from organs exhibiting low RELMα_tdTomato fluorescence intensity were electronically amplified, thus excluding any direct comparison of intensities between images of different tissues. Bars and table below summarize the absolute RELMα_tdTomato fluorescence intensities (photons/s) of the selected organs. (B) RELMα_tdTomato fluorescence analysis of the indicated organs from RetnlaCre_R26tdTomato founder mouse lines 3, 7, and 13 using the IVIS spectrum in vivo imaging system (PerkinElmer). Bar diagram on the right summarizes the fluorescence intensities of the indicated organs from each of the three founder lines as well as from a negative control sample lacking the RetnlaCre transgene (RetnlaCre7-).

Fig. S6. Flow cytometry gating strategies for the indicated cell types isolated from naïve RetnlaCre_R26tdTomato mice. Representative dot plots show gating strategies used to identify the different cell populations in this study.

Fig. S6 (continued). Flow cytometry gating strategies for the indicated cell types isolated from naïve RetnlaCre_R26tdTomato mice. Representative dot plots show gating strategies used to identify the different cell populations in this study.

Fig. S7. Analysis of RELMα_tdTomato-expressing Alv-MΦs in RetnlaCre_R26tdTomato mice. (A) Flow cytometry analysis of CD11c+ Siglec-F+ alveolar MΦs in the lungs of three representative naïve RetnlaCre_R26tdTomato mice (Cre+ #1 to #3) and one control mouse lacking the RetnlaCre transgene (Cre-). Gates labeled in red indicate percentages of the RELMα_tdTomatolow-expressing cells among total alveolar MΦs. (B) Analysis of CD11c+ Siglec-F+ alveolar MΦs in the lungs of five representative naïve bone marrow chimeras (#1 to #5) generated by transferring Ly5.1+ B6 bone marrow into irradiated Ly5.2+ RetnlaCre_R26tdTomato recipients. Gates labeled in red indicate the absence of alveolar MΦ population expressing high levels of RELMα_tdTomato reporter in these chimeras. (C) Analysis of CD11c+ Siglec-F+ alveolar MΦs in the lungs of three representative naïve bone marrow chimeras (#1 to #3) generated by transferring bone marrow from Ly5.2+ RetnlaCre_R26tdTomato donors into irradiated Ly5.1+ B6 recipient mice. Gates labeled in red indicate percentages of the RELMα_tdTomatohigh bona fide RELMα expressors among total alveolar MΦs.

Fig. S8. RELMα_tdTomato expression in MΦs derived from adoptively transferred monocytes. (A) Purity check of MACS-sorted monocytes isolated from bone marrow of RetnlaCre_R26tdTomato mice. Sorted cells consisted almost entirely of CD115+ Ly6C+ monocytes, whereas no detectable Sca-1+ c-Kit+ bone marrow progenitor cells were found. (B) Representative flow cytometry analysis of PEC cells from WT and STAT6ko recipient mice intraperitoneally transferred with monocytes isolated from RetnlaCre_R26tdTomato_WT, RetnlaCre_R26tdTomato_STAT6ko or RetnlaCre_R26tdTomato_IL-4/IL-13ko donors (as indicated) 5 days prior to analysis. (C) Ly5.1+ B6 recipients were infected with N.brasiliensis (#1 to #4) or left uninfected (naïve), and intravenously transferred with monocytes isolated from Ly5.2+ RetnlaCre_R26tdTomato mice 3 days post infection. Flow cytometry analysis of recipients’ lungs was performed at day 40 post infection. Transferred cells were detected as Ly5.2+ RELMα_tdTomato+ population, subgated for CD68+ CD11c+ MΦs and further analyzed for Siglec-F+ alveolar versus MHC-II+ interstitial MΦ populations.

Fig. S9. Analysis of MΦs in the lungs of RetnlaCre_R26tdTomato mice infected with N. brasiliensis. (A) Flow cytometry gating strategy for interstitial (Int)-MΦs as CD68+ MHC-II+ CD11b+ CD11c+ cells analyzed at day 20 post N. brasiliensis infection. Int-MΦs were further gated into RELMα_tdTomato+ cells (R1 gate) and RELMα_tdTomato− cells (R2 gate). Alveolar (Alv)-MΦs were gated as SSC-Hhi Siglec-F+ CD68+ MHC-IIdim/− CD11c+ and eosinophils as SSC-Hhi Siglec-F+ CD68– MHC-II– cells. (B) Bone marrow chimeras were generated by transferring equal amounts of bone marrow from RetnlaCre_R26tdTomato mice and IL-4/eGFP reporter mice into irradiated B6 recipients. Following 8 weeks of reconstitution, chimeras were either left untreated or infected with N.brasiliensis and analyzed 11 days post infection. Immunofluorescent analysis of the lung of chimeric mice included DRAQ5 staining for nuclei (white), CD68 staining for total lung MΦs (blue), IL-4eGFP signal (amplified via an anti-GFP antibody) indicating the IL-4 producers (green), and the endogenous RELMα_tdTomato signal indicating RELMa-producing donor-derived MΦs (red). Images on the far right show overlay of the respective channels (merge). Scale bars = 100 µm. (C) Lungs of RetnlaCre_R26tdTomato mice were isolated at day 8 post N. brasiliensis infection and Int-MΦs were sorted as CD11c+ MHC-II+ RELMα_tdTomato-expressing (black bars) and RELMα_tdTomato-negative cells (white bars) corresponding to R1 and R2 gates indicated in (A), respectively. Relative mRNA expression of the indicated M2 markers (normalized to Hprt mRNA) in sorted populations was analyzed by quantitative RT-PCR.

Fig. S10. Depletion efficiency of RELMα_tdTomato+ cells following diphtheria toxin treatment of RetnlaCre_R26tdTomato/iDTR mice. Flow cytometry analysis of F4/80+ CD11b+ MΦs in the skin (A), MHC-II+ CD11b+ interstitial MΦs in the lung (B), and MHC-II+ Ep-CAM+ CD11c- Siglec-F- type 2 pneumocytes (C) two days post intraperitoneal injection of diphtheria toxin.