Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Mar;31(3):868-881.
doi: 10.1096/fj.201600856R. Epub 2016 Nov 30.

Interleukin-10-mediated Regenerative Postnatal Tissue Repair Is Dependent on Regulation of Hyaluronan Metabolism via Fibroblast-Specific STAT3 Signaling

Affiliations
Free PMC article

Interleukin-10-mediated Regenerative Postnatal Tissue Repair Is Dependent on Regulation of Hyaluronan Metabolism via Fibroblast-Specific STAT3 Signaling

Swathi Balaji et al. FASEB J. .
Free PMC article

Abstract

The cytokine IL-10 has potent antifibrotic effects in models of adult fibrosis, but the mechanisms of action are unclear. Here, we report a novel finding that IL-10 triggers a signal transducer and activator of transcription 3 (STAT3)-dependent signaling pathway that regulates hyaluronan (HA) metabolism and drives adult fibroblasts to synthesize an HA-rich pericellular matrix, which mimics the fetal regenerative wound healing phenotype with reduced fibrosis. By using cre-lox-mediated novel, inducible, fibroblast-, keratinocyte-, and wound-specific STAT3-knockdown postnatal mice-plus syngeneic fibroblast cell-transplant models-we demonstrate that the regenerative effects of IL-10 in postnatal wounds are dependent on HA synthesis and fibroblast-specific STAT3-dependent signaling. The importance of IL-10-induced HA synthesis for regenerative wound healing is demonstrated by inhibition of HA synthesis in a murine wound model by administering 4-methylumbelliferone. Although IL-10 and STAT3 signaling were intact, the antifibrotic repair phenotype that is induced by IL-10 overexpression was abrogated in this model. Our data show a novel role for IL-10 beyond its accepted immune-regulatory mechanism. The opportunity for IL-10 to regulate a fibroblast-specific formation of a regenerative, HA-rich wound extracellular matrix may lead to the development of innovative therapies to attenuate postnatal fibrosis in organ systems or diseases in which dysregulated inflammation and HA intersect.-Balaji, S., Wang, X., King, A., Le, L. D., Bhattacharya, S. S., Moles, C. M., Butte, M. J., de Jesus Perez, V. A., Liechty, K. W., Wight, T. N., Crombleholme, T. M., Bollyky, P. L., Keswani, S. G. Interleukin-10-mediated regenerative postnatal tissue repair is dependent on regulation of hyaluronan metabolism via fibroblast-specific STAT3 signaling.

Keywords: extracellular matrix; fibrosis; inflammatory cytokines; scarless; wound healing.

Figures

Figure 1.
Figure 1.
IL-10 induces a fetal-type HA-enriched PCM in AFBs. AF) Living fibroblasts were imaged with phase contrast to visualize cell bodies, and the area occupied by the pericellular HA coat after 24 h of treatment was assessed by the exclusion of red blood cells from the cell perimeter. White and green dotted lines were artificially drawn in A to delineate the margin of the HA coat and cell body, respectively. Representative cells are shown for FFBs and AFBs under different treatment conditions: FFBs (A), FFBs treated with HYAL (B), IL-10−/− FFBs (C), AFBs (D), AFBs treated with IL-10 (E), and AFBs treated with IL-10 and HYAL (F). G) We quantified the ratio of the means ± sd of 3 experiments (20 cells analyzed per experiment). HK) Scanning electron microscopy was performed to demonstrate HA cable-like structures on the cell body outlined by green dotted line. Representative cells are shown for different cell types under different treatment conditions: FFBs (H), AFBs (I), AFBs treated with IL-10 (J), and AFBs treated with IL-10 and HYAL (K). See also Supplemental Fig. S1. Scale bar, 50 μm (AF), 5 μm (HK). **P < 0.01 by ANOVA and post hoc Bonferroni tests.
Figure 2.
Figure 2.
IL-10 induction of HA synthesis via phosphorylation and nuclear translocation of STAT3, and the differential regulation of HASs and HYALs. A) Western blots of p-STAT3 and total STAT3 in AFBs in response to IL-10 stimulation at 30 min and 1 and 2h. β-Actin was analyzed as a loading control. Densitometric analysis of means ± sd of 3 experiments is shown in the bar plots. B, C) Cells were labeled with a STAT3 Ab and the representative images, with zoomed in insets, for the perinuclear STAT3 staining in control AFB (B) as well as the nuclear translocation of activated STAT3 after AFB stimulation with IL-10 for 60 min (C) are shown. DI) Gene expression of HAS1 (D), HAS2 (E), HAS3 (F), HYAL1 (G), HAS2 (H), and KIAA1199 (I) in IL-10–stimulated AFBs at 1, 2, 3, and 6 h after stimulation with IL-10. Bar plots represent mean ± sd for 2 experiments. J) HA ELISA for AFBs 24 h after IL-10 (200 ng/ml) stimulation. Means ± sd of 3 experiments. K) Schematic illustration of the IL-10 pathway (see also Supplemental Fig. S2). Scale bar, 50 μm. *P < 0.05, **P < 0.01 by ANOVA and post hoc Bonferroni tests.
Figure 3.
Figure 3.
Competitive inhibition of IL-10 binding to IL-10R1 abrogates the effect of IL-10 on STAT3 nuclear translocation and HA-rich PCM formation. AC) Immunofluorescence staining for STAT3 translocation in AFBs in response to IL-10 with (C) or without (B) addition of an α-IL-10R1 Ab that inhibits IL-10 binding, along with control (A). Data are for 60 min after stimulation. DF) Expression of HAS1 (D), HAS2 (E), and HAS3 (F) mRNA in response to IL-10 treatment at 1, 2, 3, and 6 h in the setting of α-IL-10R1 addition. Bar plots represent means ± sd for 2 experiments. G) Particle exclusion assay assessment of PCM area of AFBs at 24 h after IL-10 (200 ng/ml) treatment in the setting of α-IL-10R1 addition. H) HA ELISA of supernatants from AFBs stimulated with IL-10 with or without α-IL-10R1 treatment. Scale bar, 50 μm. ns, not significant. **P < 0.01 by ANOVA and post hoc Bonferroni tests.
Figure 4.
Figure 4.
IL-10–induced effects on regulation of HAS1-3 and HA-rich PCM are mediated via STAT3 signaling. AC) Time course for mRNA expression of HAS1 (A), HAS2 (B), and HAS3 (C) in STAT3−/− AFBs with or without IL-10 treatment. Bar plots represent means ± sd of 2 experiments. D, E) Pericellular HA coat assessment via particle exclusion assay in STAT3−/− AFBs (D) and STAT3−/− AFBs treated with IL-10 (E). F) PCM ratio and means ± sd of 3 experiments (20 cells analyzed per experiment) are shown for STAT3−/− AFBs and STAT3−/− AFBs treated with IL-10 (200 ng/ml) for 24 h. G) HA levels in culture medium of STAT3−/− AFBs and STAT3−/− AFBs treated with IL-10 (200 ng/ml) for 24 h. Means ± sd of 3 experiments (see also Supplemental Fig. S3). Scale bar, 50 μm. ns, not significant.
Figure 5.
Figure 5.
HAS2 and HA are up-regulated in IL-10–treated in vivo wounds at 3 d postwounding, and the effect of IL-10 on regenerative wound healing in postnatal wounds is dependent on HA. AD) mRNA expression of HAS1 (A), HAS2 (B), and HAS3 (C), and HA levels (D) in homogenized pretreated skin at d 0 and wound tissue at d 1, 3, and 5 postwounding. EL) Representative histology for uninjured murine skin and murine wounds treated with lentiviral IL-10 and controls at 28 d postwounding. Images are for hematoxylin and eosin (H&E) staining (E, G, I, K; images captured with a ×4 objective; scale bar, 500 μm) and Masson’s trichrome staining (F, H, J, L; images captured with a ×40 objective; scale bar, 100 μm) of the repair tissue. M, N) Representative H&E staining (M) and trichrome staining (N) of a wound treated with lentiviral IL-10 in conjunction with 4-MU, an HAS inhibitor, at d 28 postwounding. O, P) Bar graphs show 2 elements for how we quantified scar formation and wound regeneration, including the dermal appendages (O) and scar area (P), respectively (n = 4–6 animals per group). pc, panniculus carnosus muscle layer. *P < 0.05, **P < 0.01 by ANOVA and post hoc Bonferroni tests.
Figure 6.
Figure 6.
IL-10 effects in regenerative wound healing are fibroblast dependent. AD) Hematoxylin and eosin staining of wounds in different control (A), skin-specific (B), fibroblast-specific (C), and keratinocyte-specific (D) STAT3−/− mice treated with lentiviral IL-10, all at 28 d. Images are captured with a ×4 objective. E, F) Bar graphs show 2 elements for how scar formation and wound regeneration was quantified, including the dermal appendages (E) and scar area (F), respectively (n = 4–6 animals per group). See also Supplemental Fig. S4. Scale bar, 250 μm. **P < 0.05, by ANOVA and post hoc Bonferroni tests.
Figure 7.
Figure 7.
Syngeneic dermal fibroblast transplantations recapitulate the regenerative effects of IL-10 in STAT3−/− wounds in vivo. AD) Representative image from IVIS in vivo imaging of murine untreated wounds at d 1 postwounding (A) and wounds treated with syngeneic genetically modified adult fibroblasts at d 1 (B), d 5 (C), and d 30 (D) postwounding. E, F) Hematoxylin and eosin staining of d 28 wounds in skin-specific STAT3−/− mice treated with syngeneic genetically modified AFBs, which express either GFP (E) or IL-10 (F). Images are captured with a ×4 objective. G, H) Dermal appendages (G) and scar area (H) for skin-specific STAT3−/− wounds with different AFBs transplant treatments. Arrows indicate the wounds (n = 3 animals per group). Scale bar, 500 μm. **P < 0.01 by Student’s t test.

Similar articles

See all similar articles

Cited by 15 articles

See all "Cited by" articles

References

    1. Bayat A., McGrouther D. A., Ferguson M. W. (2003) Skin scarring. BMJ 326, 88–92 - PMC - PubMed
    1. Rockey D. C., Bell P. D., Hill J. A. (2015) Fibrosis--a common pathway to organ injury and failure. N. Engl. J. Med. 372, 1138–1149 - PubMed
    1. Adzick N. S., Harrison M. R., Glick P. L., Beckstead J. H., Villa R. L., Scheuenstuhl H., Goodson W. H. III. (1985) Comparison of fetal, newborn, and adult wound healing by histologic, enzyme-histochemical, and hydroxyproline determinations. J. Pediatr. Surg. 20, 315–319 - PubMed
    1. Krummel T. M., Nelson J. M., Diegelmann R. F., Lindblad W. J., Salzberg A. M., Greenfield L. J., Cohen I. K. (1987) Fetal response to injury in the rabbit. J. Pediatr. Surg. 22, 640–644 - PubMed
    1. Longaker M. T., Whitby D. J., Adzick N. S., Crombleholme T. M., Langer J. C., Duncan B. W., Bradley S. M., Stern R., Ferguson M. W., Harrison M. R. (1990) Studies in fetal wound healing, VI. Second and early third trimester fetal wounds demonstrate rapid collagen deposition without scar formation. J. Pediatr. Surg. 25, 63–68, discussion 68–69 - PubMed

Publication types

Feedback