Figure 4. Injury-induced inflammation in the left infrapatellar fat pad reduces local Igf1 expression in the DT-impaired growth plate.

(A, A’) In situ hybridization for Igf1 in the infrapatellar fat pad (IPFP, dotted lines) of left and right P1-Pit::DTR knees, at P3 (A) and P5 (A’). (B–B’’) Immunohistochemistry for the mTORC1 readout p-S6 (top), and RNA in situ hybridization for Ihh (bottom) in left P1-Pit::DTR GPs at P3, following intraarticular injection of PBS (B), IGF1 (B’) or IGF1 combined with i.p. injection of the mTORC1 inhibitor rapamycin (B’’). (C–C’’’) Immunohistochemistry for the neutrophil marker LY6B in the IPFP (dotted lines) of left and right P1-Pit::DTR knees, at P3 (C), P5 (C’) and P2 (C’’’). The inset in (C’’’) is a 2x magnification showing that some neutrophils express TNFα in a cellular compartment. Turquoise signal= autofluorescent cells. The graph in (C’’) represents the density of LY6B+ cells in left and right P1-Pit::DTR IPFP at P3 (n = 3 mice, 3–4 sections per animal). A ratio paired t-test was used to offset the variability between absolute measurements. (D–D’’) Immunoblockade of neutrophil infiltration with NIMP-R14 antibody after DT injection (D) rescues Igf1 expression in the left IPFP (D’), as well as mTORC1 signaling in the GP, 2dpi (D’’). (E, F) Quantification of bone length at P4, expressed as Left/Right ratio, for vehicle (Veh, either PBS or IgG)-treated and IGF1- (E) or NIMP-R14-treated (F) mice (unpaired two-tailed Mann-Whitney test). See also associated Figure 4—figure supplement 1–3.

DOI: http://dx.doi.org/10.7554/eLife.27210.011

Figure 4—figure supplement 1. Characterization of the IGF signaling axis in P1-Pit::DTR mice.

(A) In situ hybridization for Igf1 in the indicated tissues of P1-Pit::DTR mice, 2dpi (metaphysis from femora, rest from tibiae). To properly compare expression domains that depend on the section level, whole slide imaging was performed on sections from five animals with a slide scanner, and representative images are shown. (B–C) Representative examples of Igf2 (B, n = 3, tibiae are shown) and Igf1r expression (C, n = 4, femora are shown) in left and right GPs and surrounding tissues of P1-Pit::DTR mice. Arrowheads in (B) point to damaged regions of the left prospective articular cartilage where Igf2 expression was somewhat diminished. (D) Immunohistochemistry for SOCS3 in the GPs and surrounding tissues of P1-Pit::DTR mice (n = 4 at P2, three at P3, tibiae are shown). Magnified views of the boxed regions are shown below the overviews.

DOI: http://dx.doi.org/10.7554/eLife.27210.012

Figure 4—figure supplement 2. IGF1 supplementation or neutrophil immunoblockade do not rescue all the signaling changes in the left RZ of P1-Pit::DTR mice.

(A, B) In situ hybridization for Fgfr3 and Lef1 in left and right P1-Pit::DTR tibiae from P3 mice treated either with IGF1 (A, n = 3) or an anti-neutrophil antibody (B, n = 4). Note that only Lef1 expression in the left GP remains consistently increased in both conditions.

DOI: http://dx.doi.org/10.7554/eLife.27210.013

Figure 4—figure supplement 3. Multiple immune cells are recruited to the infrapatellar fat pad of Pit::DTR mice, which correlates with local Igf1 downregulation.

(A–B) IHC for general macrophage markers in the IPFP of Pit::DTR mice at P4. Quantifications are shown in (A’), a ratio paired t-test was used to compare left and right macrophage density in the IPFP of Pit::DTR mice. (C) Immunostaining for the neutrophil marker LY6B and in situ hybridization for Igf1 in the IPFP of LPS-injected (left knee) WT mice. (D) Immunostaining for the neutrophil marker LY6B and in situ cell death detection (TUNEL) in the IPFP of LPS-injected (left knee) WT mice.

DOI: http://dx.doi.org/10.7554/eLife.27210.014