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. 2015 Dec 1;195(11):5296-5308.
doi: 10.4049/jimmunol.1501237. Epub 2015 Oct 30.

Myeloid Cell-Restricted Insulin/IGF-1 Receptor Deficiency Protects against Skin Inflammation

Jana Knuever #  1 , Sebastian Willenborg #  1 , Xiaolei Ding  1 , Mehmet D Akyüz  1   2 , Linda Partridge  2   3 , Carien M Niessen  1   2   4 , Jens C Brüning  2   4   5   6 , Sabine A Eming  1   2   4
Affiliations

Myeloid Cell-Restricted Insulin/IGF-1 Receptor Deficiency Protects against Skin Inflammation

Jana Knuever et al. J Immunol. .

Abstract

Myeloid cells are key regulators of tissue homeostasis and disease. Alterations in cell-autonomous insulin/IGF-1 signaling in myeloid cells have recently been implicated in the development of systemic inflammation and insulin-resistant diabetes mellitus type 2 (DM). Impaired wound healing and inflammatory skin diseases are frequent DM-associated skin pathologies, yet the underlying mechanisms are elusive. In this study, we investigated whether myeloid cell-restricted IR/IGF-1R signaling provides a pathophysiologic link between systemic insulin resistance and the development of cutaneous inflammation. Therefore, we generated mice lacking both the insulin and IGF-1 receptor in myeloid cells (IR/IGF-1R(MKO)). Whereas the kinetics of wound closure following acute skin injury was similar in control and IR/IGF-1R(MKO) mice, in two different conditions of dermatitis either induced by repetitive topical applications of the detergent SDS or by high-dose UV B radiation, IR/IGF-1R(MKO) mice were protected from inflammation, whereas controls developed severe skin dermatitis. Notably, whereas during the early phase in both inflammatory conditions the induction of epidermal proinflammatory cytokine expression was similar in control and IR/IGF-1R(MKO) mice, during the late stage, epidermal cytokine expression was sustained in controls but virtually abrogated in IR/IGF-1R(MKO) mice. This distinct kinetic of epidermal cytokine expression was paralleled by proinflammatory macrophage activation in controls and a noninflammatory phenotype in mutants. Collectively, our findings provide evidence for a proinflammatory IR/IGF-1R-dependent pathway in myeloid cells that plays a critical role in the dynamics of an epidermal-dermal cross-talk in cutaneous inflammatory responses, and may add to the mechanistic understanding of diseases associated with disturbances in myeloid cell IR/IGF-1R signaling, including DM.

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Figures

Figure 1
Figure 1. Conditional targeting of the IR and IGF-1R gene
(A) Top, scheme illustrating the IR (left) and IGF-1R (right) gene construct and the 2 loxP sites flanking exon 4 (IR, left) or exon 3 (IGF-1R, right), respectively, and the PCR fragment length shown before and after successful recombination. Bottom, PCR analysis on genomic DNA isolated from peritoneal macrophages of control (IRfl/fl/IGF-1Rfl/fl) and IR/IGF-1RMKO mice showing the deletion of the floxed region in the IR (left) and IGF-1R (right) locus in the presence of LysM-driven Cre. (B) Western blot analysis for IR (left) and IGF-1R (right) of peritoneal macrophages isolated from control or IR/IGF-1RMKO mice as indicated.
Figure 2
Figure 2. Myeloid cell-specific IR/IGF-1R deficiency protects against SDS-induced skin inflammation
(A) Macroscopic appearance of control and IR/IGF-1RMKO mice 7 days after SDS application; dashed line outlines treated area; scale bars indicate 1 cm. (B) Representative histological sections (left panel) and quantitative analysis of epidermal thickness and cells within skin lesions (right panel) at indicated time points post SDS treatment: H&E staining, Ki67 (Ki67+ cells green, counter stain propidium iodide), Gr-1 (Gr-1+ cells brown, heamatoxylin counter stain), CD68 (CD68+ cells green, counter stain DAPI); each dot represents one mouse; scale bars indicate 40 μm; he=hyperproliferative epidermis, d=dermis, sf=subcutaneous fat layer, hf=hair follicle; * p-value <0.05, ** p-value <0.01, *** p-value <0.001.
Figure 3
Figure 3. Myeloid cell-specific IR/IGF-1R deficient mice show decreased expression of inflammatory mediators in SDS-treated skin
qRT-PCR analysis of selected genes in epidermal or dermal tissues of untreated skin or at different time points after SDS treatment as indicated; each dot represents one mouse. * p-value <0.05, ** p-value <0.01, *** p-value <0.001.
Figure 4
Figure 4. IGF-1/-2 expression in SDS-treated skin
qRT-PCR analysis of IGF-1 and IGF-2 genes in epidermal or dermal tissues of untreated skin or at different time points after SDS treatment as indicated; each dot represents one mouse. * p-value <0.05, *** p-value <0.001.
Figure 5
Figure 5. Myeloid cell-specific IR/IGF-1R expression controls macrophage activation
(A) Top, representative FACS analysis of single-cell suspensions of SDS-treated skin at day 7. Bottom, 7-AAD− cells (red square) were gated and analysed for expression of CD45, CD11b, Ly6G and Ly6C as indicated and quantified. (B) qRT-PCR of selected genes in CD45+CD11b+Ly6G−Ly6Chi and CD45+CD11b+Ly6G−Ly6Clo FACS sorted cells of the SDS-treated tissue, normalized to SSClowCD11b+CD115+ blood monocytes; each dot represents one mouse. * p-value <0.05, ** p-value <0.01, *** p-value <0.001.
Figure 6
Figure 6. Myeloid cell-specific IR/IGF-1R deficiency protects from UVB-induced skin inflammation
(A) Representative macroscopic appearance (top) and histology (bottom, H&E stain) of skin 7 days after UVB irradiation; dashed line outlines irradiated area; right panel, quantification of epidermal thickness; each dot represents one mouse; scale bars indicate 1 cm. (B) Left, representative FACS analysis of single-cell suspensions of UVB-irradiated skin at day 7; right, 7-AAD− cells (red square) were gated and analysed for expression of CD45, CD11b, F4/80 and quantified. (C) qRT-PCR of selected genes in CD45+CD11b+F4/80+ FACS sorted cells of the UVB-irradiated tissue, normalized to SSClowCD11b+CD115+ blood monocytes; each dot represents one mouse. * p-value <0.05, ** p-value <0.01, *** p-value <0.001.
Figure 7
Figure 7. Insulin and IGF-1 induce a pro-inflammatory macrophage phenotype in vitro associated with activation of Akt and p38α MAPK
(A) qRT-PCR analysis of selected genes in peritoneal macrophages cultured in the presence of a combination of rInsulin and rIGF-1 (+) or vehicle (−) for 3 h; gene expression in rInsulin/rIGF-1 stimulated cells is normalized to vehicle treated cells; each dot represents macrophages isolated from one mouse; * p-value <0.05, *** p-value <0.001. (B) PathScan® Intracellular Signaling Array Kit analysis of peritoneal macrophages isolated from control and IR/IGF1RMKO mice cultured for 15 min in rInsulin/rIGF-1 or vehicle; presented is the difference of chemiluminescent intensity of selected phosphorylated proteins between rInsulin/rIGF-1 stimulated or vehicle treated cells; presented is the mean of 2 independent experiments. (C) Western blot analysis for p-Akt and p-p38α of peritoneal macrophages, stimulated for indicated time periods with rInsulin/rIGF-1; C=control mice, Ko=IR/IGF-1RMKO mice; α-Tubulin and GAPDH served as loading controls.
Figure 8
Figure 8. Hypothetical model of myeloid cell-restricted IR/IGF-1R function in the dynamics of the epidermal-dermal crosstalk in skin inflammation
Epidermal injury causes rapid release of pro-inflammatory mediators (IL-6, IL-1β, TNFα) and chemokines (MIP1α, MCP1, MCP3) that mediate effective recruitment of blood monocytes/macrophages into the dermis; influx of myeloid cells during the early phase of inflammation appears independent of myeloid cell-restricted IR/IGF1-R; during late stage inflammation, myeloid cell-restricted IR/IGF1-R activation induces a pro-inflammatory macrophage phenotype that sustains epidermal inflammation by the release of pro-inflammatory mediators (IL-6, IL-1β, TNFα).
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