Available via license: CC BY 4.0
Content may be subject to copyright.
Supplementary Material
SUPPLEMENTARY FIGURES AND TABLES 1
Supplementary Figures 2
Supplementary Figure 1 | Flow cytometry gating strategy for ILC2s. (A) Lung and (B) kidney single 3
cell suspensions from Il5venus/+Il13td-tomato/+ mice, n=8, were gated for ILC2s (CD45+Lineage-[TCR-
4
[TCRαβ-TCRγδ-CD8-CD4-]CD11b-GR-1-B220-TER-119-CD3-NK-1.1-]IL-5
7R+CD90.2+ST2+FSClowSSClow single cells). (C) Representative FACS plots of IL-5 and (D) IL-13 6
expression in kidney ILC2s. (E) Gating strategy demonstrating the proportion of CD45+CD19-CD11b-
7
CD49b-CD90.2+IL-5+ single cells which were consistent with (F) ILC2s (CD3-CD4-) and TH2s 8
(CD3+CD4+) in kidney single cell suspensions from Il5td-tomatoCre; Rosa-CAG-RFP mice, n=8. 9
Supplementary Figure 2 | Immunofluorescence imaging of IL-5+ cells in the kidney. Kidney sections 10
from Il5td-tomatoCre; Rosa26-CAG-RFP mice were stained with DAPI, α-SMA and DsRed to visualize 11
nuclei, smooth muscle actin and IL-5, respectively. (A) Visualization of broad regions of the mouse 12
kidney, IL-5+ staining was identified in the calyx, capsule, cortex and medulla. (B) IL-5+ staining was 13
localized to blood vessels in the kidney. DsRed identified IL-5+ cells and imaris software identified 14
maximum projection surfaces and enhanced the pixel area for ease of visualization. 15
Supplementary Figure 3 | Histopathology following IRI was unaltered by the reduction of ILC2s. 16
C57BL/6JAusB wild-type (WT, n=8) mice were subjected to 29-minute unilateral IRI with 17
contralateral nephrectomy and were assessed at 1, 3 and 7-days post-surgery. WT, vehicle (saline-18
treated Icosdtr/+Cd4cre/+, n=5), ILC2-reduced (Rorafl/+Il7rcre/+, n=4), ILC2-deficient (Rorafl/flIl7rcre/+, 19
n=4) and ILC2-depleted (diphtheria toxoid-treated Icosdtr/+Cd4cre/+, n=5) were assessed 7-days post-20
surgery. (A) Representative images of periodic acid-Schiff and (B) Masson’s trichrome stained kidney 21
sections following sham and IRI surgery, blue staining indicates collagen. Scale bar in each image 22
indicates 100μm. 23
Supplementary Figure 4 | IRI increased mRNA expression and histopathological score. 24
(A) Extracellular Matrix & Adhesion Molecules RT² Profiler PCR Array, truncated to 25/84 targets, 25
which were increased >2 fold in kidney homogenates from C57BL/6JAusB wild-type (WT, n=8) on 26
day 1, 3 or 7 following IRI (n=8 per timepoint), compared to the average expression across each 27
timepoint following sham surgery (n=24). (B) Semi-quantitative tubular injury score indicating injury 28
in terms of the proportion of tubules effected by casts, dilation, apoptosis and/or loss of brush border, 29
where a score of 5 indicates 76-100% of tubules were affected. (C-K) mRNA expression of injury 30
(Lcn2), inflammatory (Cxcl1, Cxcl2 and Tnf), extracellular matrix (Col1a1 and Fn1), ILC2- and M2-31
macrophage associated factors (Areg, Arg1 and Mrc1) in kidney homogenates relative to Hprt. All data 32
are expressed as mean ±SEM. * P<0.05, ns not significant; by Mann-Whitney U-test. 33
Supplementary Material
2
Supplementary Tables 34
Supplementary Table 1 35
Panel 1: ILC2
Antigen Product code Clone Fluorophore Dilution factor Source
CD278 (ICOS) 565886 C398.4A BV421 100 BD Biosciences
TCR-αβ 563221 H57-597 BV510 300 BD Biosciences
TCR-γδ 563218 GL3 BV510 300 BD Biosciences
CD4 563106 RM4-5 BV510 300 BD Biosciences
CD8a 563068 53-6.7 BV510 300 BD Biosciences
KLRG1 740553 2F1 BV650 100 BD Biosciences
CD45 550994 30-F11 PerCP-Cy5.5 200 BD Biosciences
CD127 (IL-7Rα) 562419 SB/199 PE-CF594 100 BD Biosciences
CD25 552880 PC61 PE-Cy7 50 BD Biosciences
IL-33R (ST2) 17-9335-82 RMST2-2 APC 50 eBioscience
CD11b 557960 M1/70 AF700 100 BD Biosciences
LY-6G/C (GR-1) 557979 RB6-8C5 AF700 100 BD Biosciences
CD45R (B220) 557957 RA3-6B2 AF700 100 BD Biosciences
TER-119 560508 TER-119 AF700 100 BD Biosciences
CD3 561388 17A2 AF700 100 BD Biosciences
NK-1.1 560515 PK136 AF700 100 BD Biosciences
3
CD90.2 561641 53-2.1 APC-Cy7 200 BD Biosciences
IL-5 - - Venus (BB515) - Reporter mice
IL-13 - - Td-tomato (PE) - Reporter mice
Panel 2: ILC2 and TH2
Antigen Product code Clone Fluorophore Dilution factor Source
CD45 564279 30-F11 BUV395 400 BD Biosciences
CD90.2 140318 53-2.1 BV605 400 Biolegend
CD3 100236 17A2 APC 100 Biolegend
CD4 100557 RM4-5 BV711 200 Biolegend
CD8a 100750 53-6.7 BV785 200 Biolegend
CD49b 108918 DX5 Pacific Blue 300 Biolegend
CD19 115520 6D5 PE-Cy7 200 Biolegend
CD11b 101259 M1/70 BV650 200 Biolegend
IL-5 - - Td-tomato (PE) - Reporter mice
Supplementary Table 1 | Flow cytometry antibodies for assessing group 2 innate lymphoid cells 36
(ILC2s; Panel 1) alone, or in conjunction with T helper type 2 cells (TH2; Panel 2). 37
Supplementary Table 2 38
Gene ID Forward primer (5`-3`) Reverse primer (5`-3`)
Areg TTGCTGCTGGTCTTAGGCTC TGGTCCCCAGAAAGCGATTC
Arg1 GGCAGAGGTCCAGAAGAATG GTGAGCATCCACCCAAATG
Ccl20 AGACGCCTCTTCCTTCGAGAGC TGCTTTGGATCAGCGCACACA
Supplementary Material
4
Ccl5 TGGCTCGGACACTCCCTG GGGTTGGCACACACTTGGCGG
Col1a1 CTTCACCTACAGCACCCTTGTG TGACTGTCTTGCCCCAAGTTC
Cxcl1 GCTGGGATTCACCTCAAGAA CTTGGGGACACCTTTTAGCA
Cxcl10 CCAAGTGCTGCCGTCATTTTC TCCCTATGGCCCTCATTCTCA
Cxcl2 TGCTGCTGGCCACCAACCAC AGTGTGACGCCCCCAGGACC
Fn1 GCACTGCTGCTGATTCAAGTTC AGTTGCTCCTGGCTGGTATG
Hprt AGGCCAGACTTTGTTGGATTTGAA CAACTTGCGCTCATCTTAGGCTTT
Lcn2 TACAATGTCACCTCCATCCTGG CCACTTGCACATTGTAGCTCT
Mrc1 CATGGATGTTGATGGCTACTGG CCATAGAAAGGAATCCACGCAGT
Nos2 AGCGAGGAGCAGGTGGAAGACT CCATAGGAAAAGACTGCACCGAA
Tnf TCTGTCTACTGAACTTCGGGGTGA TTGTCTTTGAGATCCATGCCGTT
Supplementary Table 2 | qPCR primer sequences. 39