Analysis of combinatorial chemokine receptor expression dynamics using multi-receptor reporter mice
- Chemokine Research Group, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, United Kingdom
- Center for Molecular and Cellular Bioengineering, Biotechnology Center, Technische Universität Dresden, Germany
- Shandong University–Helmholtz Institute of Biotechnology, State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, China
- Max-Planck-Institute for Cell Biology and Genetics, Germany
Figures
Figure 1 with 3 supplements
Generation of the reporter mice.
(A) Reporters were selected for this study based on their discrete excitation and emission spectra. (B) (i) The inflammatory Ccr genes were targeted in a bacterial artificial chromosome (BAC). (ii) …
Figure 1—figure supplement 1
Gating strategies used in the study.
Gating strategies used for the isolation of cell subsets in (A) spleen, (B) bone marrow and blood, and (C) air-pouch membrane. Abbreviations are: Eϕ, eosinophils; Nϕ, neutrophils; Mϕ, macrophages.
Figure 1—figure supplement 2
Gating strategies used in the study.
Gating strategies used for the isolation of (A) lung and (B) kidney cell subsets. Abbreviations are: Nϕ, neutrophils; Eϕ, eosinophils; Alv Mϕ, alveolar macrophages; Mϕ, macrophages; moDCs, …
Figure 1—figure supplement 3
Normal leukocyte recruitment in the reporter mice.
Leukocyte counts determined by flow cytometry in resting (i) bone marrow, (ii) lung, (iii) blood, (iv) spleen, and (v) kidney. Data are shown as mean ± SEM and are compiled from at least two …
Figure 2
Inflammatory CC chemokine receptor (iCCR) reporter expression accurately mirrors iCCR surface presentation.
(A) (i) Flow cytometric analysis of CCR2 antibody binding and mRuby2 expression in spleen Ly6Chi monocytes at rest. (ii) Quantification of the percentage of Ly6Chi monocytes binding CCR2 antibody …
Figure 3
Inflammatory CC chemokine receptor (iCCR) expression in bone marrow and blood leukocytes at rest.
(A) Flow cytometric analysis of Clover/CCR1 and mRuby2/CCR2 expression in (i) bone marrow and (iii) circulating Ly6Chi monocytes. Quantification of the percentage of Ly6Chi monocytes expressing the …
Figure 4 with 4 supplements
Inflammatory CC chemokine receptor (iCCR) expression in resting lung.
(A) (i) Flow cytometric analysis of mRuby2/CCR2 expression in Ly6Chi monocytes. (ii) Quantification of the percentage of Ly6Chi monocytes expressing the iCCR reporters. (iii) Distribution of the …
Figure 4—figure supplement 1
Reporter co-expression in resting tissues.
Figure 4—figure supplement 2
Inflammatory CC chemokine receptor (iCCR) expression in resting lung.
(A) Flow cytometric analysis of (i) Clover/CCR1, (ii) mRuby2/CCR2, and (iii) iRFP682/CCR5 expression in F4/80Lo monocyte-derived dendritic cells (moDCs) in resting lung. (iv) Quantification of the …
Figure 4—figure supplement 3
Inflammatory CC chemokine receptor (iCCR) expression in monocytes and macrophages during differentiation.
(A) Schematic of the procedure used to transfer monocytes into the air-pouch. (B) iCCR expression on REP Ly6CHi monocytes immediately before injection into the air-pouch. (C) iCCR expression on …
Figure 4—figure supplement 4
Inflammatory CC chemokine receptor (iCCR) reporters are readily visualised in tissues.
Spleens from resting mice were isolated and imaged using an AxioImager M2 microscope (Zeiss). Different magnifications are shown.
Figure 5 with 2 supplements
Inflammatory CC chemokine receptor (iCCR) expression in resting kidney.
(A) (i) Flow cytometric analysis of mRuby2/CCR2 expression in Ly6Chi monocytes. (ii) Quantification of the percentage of Ly6Chi monocytes expressing the iCCR reporters. (iii) Distribution of the …
Figure 5—figure supplement 1
Reporter co-expression in resting tissues.
Figure 5—figure supplement 2
Inflammatory CC chemokine receptor (iCCR) reporters visualised in resting kidney.
Kidneys from resting mice were isolated and imaged using an AxioImager M2 microscope (Zeiss). Inlet shows a myeloid cell co-expressing Clover/CCR1 and iRFP682/CCR5.
Figure 6
Inflammatory CC chemokine receptor (iCCR) expression in acutely inflamed bone marrow and blood leukocytes.
(A) Schematic of the procedure used to induce acute inflammation using the air-pouch model. (B) Flow cytometric analysis of Clover/CCR1 and mRuby2/CCR2 expression in (i) bone marrow and (iv) …
Figure 7
Inflammatory CC chemokine receptor (iCCR) expression in bone marrow and blood under sustained inflammation.
(A) Schematic of the procedure used to induce sustained inflammation using interferon gamma (IFNγ) release from osmotic pumps. (B) Flow cytometric analysis of Clover/CCR1 expression in (i) bone …
Figure 8 with 2 supplements
Inflammatory CC chemokine receptor (iCCR) expression in inflamed tissues: the air-pouch model.
(A) Flow cytometric analysis of Clover/CCR1, mRuby2/CCR2, and iRFP682/CCR5 expression in (i) Ly6Chi monocytes and (iii) F4/80+ macrophages isolated from the inflamed air-pouch. Quantification of …
Figure 8—figure supplement 1
Reporter co-expression in inflamed air-pouch.
Figure 8—figure supplement 2
Inflammatory CC chemokine receptor (iCCR) expression in inflamed air-pouch: lymphoid cells.
(A) Flow cytometric analysis of (i) mRuby2/CCR2 and (ii) iRFP682/CCR5 expression in CD11b- NK cells. (iii) Quantification of the percentage of CD11b- NK cells expressing the iCCR reporters. (iv) …
Figure 9 with 2 supplements
Inflammatory CC chemokine receptor (iCCR) expression in inflamed tissues: the intranasal lipopolysaccharide (LPS) model.
(A) (i) Schematic of the procedure used to induce acute lung inflammation using intranasal administration of LPS. (ii) Quantification of monocyte, macrophage, alveolar macrophage, and eosinophil …
Figure 9—figure supplement 1
Inflammatory CC chemokine receptor (iCCR) reporters visualised in resting and inflamed lungs.
Lungs from PBS- (A) or LPS- (B) treated mice were isolated and imaged using an AxioImager M2 microscope (Zeiss).
Figure 9—figure supplement 2
Reporter co-expression in inflamed lung.
Figure 10
Proposed model for the contribution of the inflammatory CC chemokine receptors (iCCRs) to leukocyte migration.
Videos
Video 1
Real-time in vivo imaging of iCCR reporters in resting mammary gland.
Tables
Table 1
Primers used in the study.
| A. Detection of the iCCR reporters | |
|---|---|
| iRFP682 QPCR1 | GTCACCCCAGACCTCAATCC |
| iRFP682 QPCR2 | AACGATCAATCCCCACAGTC |
| mRuby2 RT F | TGGGAAAGAGTTACGAGATACGA |
| mRuby2 RT R | AACGAGACAGCCATCCTCAA |
| Clover QPCR1 | AACGGCATCAAGGCTAACTTC |
| Clover QPCR2 | GGGTGTTCTGCTGGTAGTGG |
| mTagBFP2 QPCR1 | ACCGTGGACAACCATCACTT |
| mTagBFP2 QPCR2 | CCTCGACCACCTTGATTCTC |
| B. Quantification of iCCR-REP cluster insertion | |
| CCR1prom QPCR1 | TCAACTCAACTCCATCCAACC |
| CCR1prom QPCR2 | CTGTCTTTCCTCTCTGCTCCA |
| BAC-CPN1 Stan1 | CAGCTAGCCCCCAGGTGACA |
| BAC-CPN1 Stan2 | AGTCTTTCTTTCCTGCGTTGTATG |
| BAC-CPN1 QPCR1 | GATAAAGGGAAGCAGACACCAG |
| BAC-CPN1 QPCR2 | CAGCAGGGAGGAAAGAAGAGT |
| BAC-CPN2 Stan1 | AGTCTTTCTTTCCTGCGTTGTATG |
| BAC-CPN2 Stan2 | AAAACCAGACAGGATAGATAACTG |
| BAC-CPN2 QPCR1 | AGGGGTGGAAGCCTATCTCTAC |
| BAC-CPN2 QPCR2 | TGGCAGCATTTACAGGGTCT |
| BAC-CPN3 Stan1 | GGATGGGAGGGAATTTGGAGAAGA |
| BAC-CPN3 Stan2 | GCTTTGTGAAGGCCGAGGTCTAA |
| BAC-CPN3 QPCR1 | CCCCATCCATAACACAAACC |
| BAC-CPN3 QPCR2 | CAAAATGAGCACCTCCCTTC |
| CCR2exon Stan1 | AGGGAGAGCAGAAGGCTAA |
| CCR2exon Stan2 | CCCAGGAAGAGGTTGAGAGA |
| CCR2exon QPCR1 | TGTGGGACAGAGGAAGTGG |
| CCR2exon QPCR2 | GGAGGCAGAAAATAGCAGCA |
| CCR5exon Stan1 | ACCCATTGAGGAAACAGCAA |
| CCR5exon Stan2 | CTTCTGAGGGGCACAACAAC |
| CCR5exon QPCR1 | TTTGTTCCTGCCTTCAGACC |
| CCR5exon QPCR2 | TTGGTGCTCTTTCCTCATCTC |
| TBP Stan1 | GAGTTGCTTGCTCTGTGCTG |
| TBP Stan2 | ATACTGGGAAGGCGGAATGT |
| TBP QPCR1 | TGCTGTTGGTGATTGTTGGT |
| TBP QPCR2 | AACTGGCTTGTGTGGGAAAG |
Table 2
Antibodies used in the study.
| Antibody | Clone | Source | Working dilution |
|---|---|---|---|
| Anti-mouse CD45 | 30-F11 | eBioscience | 1/100 |
| Anti-mouse CD11b | M1/70 | eBioscience | 1/100 |
| Anti-mouse SiglecF | E50-2440 | BD Bioscience | 1/100 |
| Anti-mouse F4/80 | BM8 | eBioscience | 1/100 |
| Anti-mouse CD64 | X54-5/7.1 | BD Bioscience | 1/100 |
| Anti-mouse Ly6C | HK1.4 | BioLegend | 1/100 |
| Anti-mouse CD11c | HL3 | BD Bioscience | 1/100 |
| Anti-mouse MHCII | M5/114.15.2 | BioLegend | 1/100 |
| Anti-mouse Ly6G | 1A8 | BD Bioscience | 1/100 |
| Anti-mouse CD19 | eBio1D3 (1D3) | eBioscience | 1/100 |
| Anti-mouse CCR2 | SA203G11 | BioLegend | 1/50 |
| Anti-mouse CCR3 | J073E5 | BioLegend | 1/50 |
| Anti-mouse CCR5 | HM-CCR5 (7A4) | eBioscience | 1/50 |
