Skin Wounding-Induced Monocyte Expansion in Mice Is Not Abrogated by IL-1 Receptor 1 Deficiency

Abstract

The aim of this study was to determine whether skin wounding induces monocyte (Mo) expansion in bone marrow and whether IL-1R1 signaling regulates this process. Our data show that skin wounding increases myeloid lineage-committed multipotent progenitors (MPP3 subset) and Mo in bone marrow, but this expansion is not impaired in Il1r1^-/- mice. We also demonstrate that M-CSF-induced differentiation of myeloid progenitors into Mo is not impaired by the loss of IL-1R1 ex vivo, indicating that IL-R1 deficiency does not abrogate myeloid progenitor differentiation potential. In addition, we observed modestly delayed wound closure in Il1r1^-/- mice associated with higher frequency of Ly6C^lo Mo in the circulation at baseline and in wounds early after injury. Thus, in contrast to other models of inflammation that involve IL-1R1-dependent monopoiesis, our results demonstrate that skin wounding induces Mo progenitor and Mo expansion independently of IL-1R1 signaling.

Figure 1:

Skin wounding-induced expansion of MPP3 compartment is not affected by IL-1R1 signaling deficiency. (A) Representative flow-cytograms showing gating strategy for flow-cytometry analysis of HSPC subsets in BM. (B-E) Percentage (of total BM cells (BMC); upper panels) and number (lower panels) of LT-HSC (Lin-Sca-1+cKit+(LSK)Flk2-CD48-CD150+), ST-HSC (LSK Flk2-CD48-CD150-), MPP3 (LSK Flk2-CD48+CD150-) and MPP4 (LSK Flk2+CD150-) in BM. (F) Gating strategy for MyP subsets and LSK in BM. (G-J) Percentage (of total BMC; upper panels) and number (lower panels) of CMP (Lin-Sca-1-cKit+FcRγloCD34+), GMP (Lin-Sca-1-cKit+FcRγhiCD34+), MyP (Lin-Sca-1-cKit+) and LSK in BM. Results are represented as mean ± SD, n=7 mice for each strain and each time point. a, mean value significantly different from day 0 in WT; b, mean value significantly different from day 0 in Il-1r1^−/−. p≤0.05 is significant.

Figure 2:

Skin wounding increases GMP proliferation IL-1R1 independently. Representative flow-cytograms showing BrdU+ cells gated on MPP3 (A), GMP (B), HSC (C) and CMP (D) (left panels) and percentage of BrdU+ cells in the corresponding cell populations (right panels). Results are represented as mean ± SD, n=3 to 4 mice for each strain and each time point. *p≤0.05. (E) IL-1β concentration in BM plasma. Results are represented as mean ± SD, n=4 to 9 mice for each time point.

Figure 3:

IL-1R1 deficiency does not alter skin wounding-induced monocyte expansion in BM. (A) Representative flow-cytograms showing gating strategy for flow-cytometry analysis of Mo subsets in BM. (B-D) Percentage (of total BMC; upper panels) and number (lower panels) of total Mo (Ly6G-CD11b+CD115+), Ly6Chi Mo (Ly6G-CD11b+CD115+Ly6Chi) and Ly6Clo Mo (Ly6G-CD11b+CD115+Ly6Clo) in BM. Results are represented as mean ± SD, n=7 mice for each strain and each time point. a, mean value significantly different from day 0 in WT; b, mean value significantly different from day 0 in Il-1r1^−/−. (E) Representative flow-cytograms showing BrdU+ cells gated on Ly6Chi (left panel) and percentage of Ly6Chi BrdU+ cells (right panel). Results are represented as mean ± SD, n=3 to 4 mice for each strain and each time point. *p≤0.05.

Figure 4:

IL-1R1 signaling deficiency does not abrogate expansion potential of MyP. (A) Experimental scheme. (B) Representative microscopic images of MyP cultures after 4 days of incubation. Images taken at 10x magnification. (C) Representative flow-cytograms for flow-cytometry analysis of CD11bhiLy6Chi Mo after 4 days of culture. Number of total cells (D) and CD11bhiLy6Chi Mo (E) after 4 days of culture. Results are represented as mean ± SD. BM cells were collected from 2 mice for each strain and experiment was done once in triplicates. *p≤0.05, ***p≤0.001, ****p≤0.0001.

Figure 5:

Frequency of Ly6Clo Mo is higher in the circulation of Il1r1^−/− mice at steady state. (A) Representative flow-cytograms showing gating strategy for flow-cytometry analysis of Mo subsets in peripheral blood. (B-D) Percentage (of total BMC; upper panels) and number (lower panels) of total Mo (Ly6G-CD11b+CD115+), Ly6Chi Mo (Ly6G-CD11b+CD115+Ly6Chi) and Ly6Clo Mo (Ly6G-CD11b+CD115+Ly6Clo) in peripheral blood. Results are represented as mean ± SD, n=3 mice for each strain and each time point. c, mean value significantly different between two strains at same time point. p≤0.05 is significant.

Figure 6:

IL-1R1 signaling deficiency increases Ly6Clo Mo during early wound repair. (A) Percentage of wound area as compared with day 0 wounds. ImageJ software was used to measure wound area. (B) Representative flow-cytograms showing gating strategy for flow-cytometry analysis of innate immune cells at wounds. (C-H) Percentage (of live cells; upper panels) and number (lower panels) of total leukocytes (CD45+), Mo/Mp (CD45+Ly6G-CD11b+), Ly6Chi Mo (CD45+Ly6G-CD11b+F4/80-Ly6Chi), Ly6Clo Mo (CD45+Ly6G-CD11b+F4/80-Ly6Clo), Ly6Chi Mp (CD45+Ly6G-CD11b+F4/80+Ly6Chi) and Ly6Clo Mp, (CD45+Ly6G-CD11b+F4/80+Ly6Clo) in the wounds. Results are represented as mean ± SD, n=7 mice for each strain and each time point. a, mean value significantly different from day 0 in WT; b, mean value significantly different from day 0 in Il1r1^−/−; c, mean value significantly different between two strains at same time point. p≤0.05 is significant.