Oxidant therapy improves adipogenic differentiation of adipose-derived stem cells in human wound healing

Christian Ploner,Karin Joehrer,Doris Wilflingseder,Gerhard Pierer,Christof Seifarth,Susanne Lobenwein,Catherine E Connolly,Giuseppe Cappellano,Evi M Morandi,Markus Nagl,Martin Hermann,Tina Rauchenwald,Johannes Rainer

doi:10.1186/s13287-021-02336-3

Abstract

BackgroundAdipose-derived stem cells (ASC) and adipocytes are involved in numerous physiological and pathophysiological conditions, which have been extensively described in subcutaneous and visceral fat depots over the past two decades. However, much less is known about ASC and adipocytes outside classical fat tissue depots and their necessity in tissue remodeling after injury. Therefore, we investigated the etiology of adipocytes in human granulation tissue and define their possible role wound healing.MethodsIdentification of human wound tissue adipocytes was determined by immunohistochemical staining of granulation tissue sections from patients undergoing surgical debridement. Stromal cell fractions from granulation tissue and subcutaneous fat tissue were generated by collagenase type II-based protocols. Pro- and anti-inflammatory wound bed conditions were mimicked by THP1- and CD14+ monocyte-derived macrophage models in vitro. Effects of macrophage secretome on ASC differentiation and metabolism were determined by immunoblotting, flow cytometry, and microscopy assessing early and late adipocyte differentiation states. Functional rescuing experiments were conducted by lentiviral transduction of wildtype PPARG, IL1RA, and N-chlorotaurine (NCT) treatment.ResultsSingle and clustered adipocyte populations were detected in 11 out of 13 granulation tissue specimens and single-cell suspensions from granulation tissue showed adipogenic differentiation potential. Pro-inflammatory signaling by IFNG/LPS-stimulated macrophages (M (IFNG/LPS)) inhibited the maturation of lipid droplets in differentiated ASC. In contrast, anti-inflammatory IL4/IL13-activated macrophages (M (IL4/IL13)) revealed minor effects on adipocyte development. The M (IFNG/LPS)-induced phenotype was associated with a switch from endogenous fatty acid synthesis to glycolysis-dominated cell metabolism and increased pro-inflammatory cytokine production. Impaired adipogenesis was associated with increased, but seemingly non-functional, CEBPB levels, which failed to induce downstream PPARG and CEBPA. Neither transgenic PPARG overexpression, nor inhibition of IL1B was sufficient to rescue the anti-adipogenic effects induced by IFNG/LPS-activated macrophages. Instead, macrophage co-treatment during stimulation with NCT, a mild oxidant produced by activated granulocytes present in human wounds in vivo, significantly attenuated the anti-adipogenic effects.ConclusionsIn conclusion, the appearance of adipocytes in wound tissue indicates a prevailing anti-inflammatory environment that could be promoted by NCT treatment and may be associated with improved healing outcomes.Graphical abstract

Highlights

Adipose-derived stem cells (ASC) and adipocytes are involved in numerous physiological and pathophysiological conditions, which have been extensively described in subcutaneous and visceral fat depots over the past two decades
Neither transgenic peroxisome proliferatoractivated receptor gamma (PPARG) overexpression, nor inhibition of IL1B was sufficient to rescue the anti-adipogenic effects induced by IFNG/LPS-activated macrophages
These adipocytes were clearly separated from subcutaneous fat tissue by connective tissue and embedded in a hyaluronan and polysaccharide-enriched extracellular matrix (ECM) as visualized with fluorescence labeled wheat germ agglutinin WGA555 (Fig. 1b)

Summary

Introduction

Adipose-derived stem cells (ASC) and adipocytes are involved in numerous physiological and pathophysiological conditions, which have been extensively described in subcutaneous and visceral fat depots over the past two decades. We investigated the etiology of adipocytes in human granulation tissue and define their possible role wound healing. Autologous fat grafting enhanced healing outcomes of therapy-resistant chronic wounds of varying etiologies [3]. Injectable formulations of adipose-derived stem cells (ASC) promise therapeutic and regenerative capacity in a range of tissue and disease settings [4, 5]. These findings underline the significance of adipose tissue and its main cellular constituents for wound healing and suggest their role as highly dynamic and plastic participants in inflammatory signaling environments

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