Supplementary Data

• Supplementary Figure 1 - Structure of the ‘floxed’ and MKO Hsd11b1 alleles. (A) Structure of the floxed Hsd11b1 (Hsd11b1^f) allele: exons are indicated by boxes. Blue hatched boxes indicate untranslated regions, with solid boxes representing the 11β-HSD1 open reading frame. Lower case letters indicate positions of primers used for genotyping. FRT recombination was used to remove a selection cassette, leaving a single FRT element (blue triangle). In Hsd11b1^MKO mice, Cre-mediated recombination between LoxP sites (red triangles) removes exon 3 and generates a frameshift affecting the remaining exons (red hatched boxes). (B-D) show representative genotyping PCR reactions carried out on the same 15 mice, with controls containing wild-type (WT) or Hsd11b1^f/+Cre+ (+) DNA. (B) Cre genotyping generates a 465bp product (arrow: mice 2, 5, 6, 7, 9, 11 and 13 are Cre+). (C) PCR with primers a and b, flanking the 3’ LoxP site, produces a 255bp product from the wild-type Hsd11b1 allele (arrow: WT Hsd11b1) and a 385bp product from the ‘floxed’ Hsd11b1^f allele (arrow: Floxed Hsd11b1). As expected, none of the mice carry the WT allele. (D) Primers a and c produce a 363bp product from the Hsd11b1^MKO allele (arrow; Deleted Hsd11b1), present in all Cre⁺ mice, and a 1066bp product from the floxed Hsd11b1^f allele (arrow; Floxed Hsd11b1), present in all Cre^- mice (PDF 1597 KB)
• Supplementary Figure 2 - Hsd11b1 mRNA levels are reduced in Hsd11b1^MKO mice in cells elicited to the peritoneum by thioglycollate injection. Hsd11b1^MKO (MKO: white bars) and control Hsd11b1^f/f mice (Con: black bars) were injected (i.p.) with 0.2ml 10% thioglycollate (TG) and peritoneal cells harvested. Hsd11b1 mRNA levels were measured by qPCR in (A) cells lavaged 96h after TG injection, (B) cells lavaged 24h after TG injection and (C) neutrophils, affinity purified using Ly6G antibody from cells harvested 24h after thioglycollate injection. Hsd11b1 mRNA levels are expressed relative to levels of Tbp mRNA, used as internal control. (A, B) Data are means ± SEM and were analysed by unpaired t-test; ** p<0.01, n=4-6/group). For (C), cells were pooled from 3-4 mice and values are the mean of 2 (Hsd11b1^MKO) or 3 (Hsd11b1^f/f mice) replicate samples. (PDF 2132 KB)
• Supplementary Figure 3 - A modest decrease in 11β-HSD1 protein levels in myeloid cells elicited to the peritoneum of Hsd11b1^MKO mice by thioglycollate. Western blotting was used to measure 11β-HSD1 protein levels in myeloid cells from Hsd11b1^MKO (MKO: white bars) and control Hsd11b1^f/f mice (Con: black bars). Quantification of 11β-HSD1 protein levels in (A) Peritoneal macrophages lavaged 96h after thioglycollate injection, measured relative to levels of β-tubulin, (B) Total peritoneal cells lavaged 24h after thioglycollate injection, measured relative to levels of β-tubulin, (C) Ly6G-affinity purified neutrophils isolated from cells elicited to the peritoneum 24h after thioglycollate injection, measured relative to levels of GAPDH and (D) Resident peritoneal cells (1 mouse sample/lane; note – only 3 lanes were loaded; bands in other lanes are due to spill-over). Data are means ± SEM and were analysed by unpaired t-test; *p<0.05, ***p<0.001, n=4/group (A, B) or 3/group (C). (PDF 2877 KB)
• Supplementary Figure 4 - Expression of inflammation-related genes is largely unchanged in sponges recovered from Hsd11b1^MKO mice, compared to littermate controls. RNA was extracted from sponges removed 21 days after implantation and qPCR was used to measure levels of Vegfa, Icam1, Il6, Tnfa, Ifng,Ccl5 and Cd68 mRNA, relative to Hprt mRNA, used as an internal standard. Values are in arbitrary units (AU) and are means ± SEM. Data from Hsd11b1^MKO (white bars) and Hsd11b1^f/f mice (black bars) were analysed by unpaired t test, n=6-11, *p<0.05. (PDF 1772 KB)
• Supplementary Table 1 - qPCR primers and probes (PDF 107 KB)