Targeting inflammatory macrophages with hyaluronan tetrasaccharide: effects on fibroblast collagen degradation and synthesis

Hyaluronan (HA) provides moisturizing benefits and exhibits unique biological activities based on its molecular weight. While the anti-inflammatory effects of high-molecular-weight HA have been well studied, the impact of hyaluronan tetrasaccharide (HA4), an ultralow-molecular-weight HA, on the skin...

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Main Authors: Eiko Uno, Florence Kim, Mihoko Yoshino, Yasunari Sato, Masao Hashimoto, Kenji Watanabe, Yoichi Mizukami, Jun Muto
Format: Article
Language:English
Published: Frontiers Media S.A. 2025-06-01
Series:Frontiers in Immunology
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Online Access:https://www.frontiersin.org/articles/10.3389/fimmu.2025.1592751/full
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author Eiko Uno
Florence Kim
Mihoko Yoshino
Yasunari Sato
Masao Hashimoto
Kenji Watanabe
Yoichi Mizukami
Jun Muto
author_facet Eiko Uno
Florence Kim
Mihoko Yoshino
Yasunari Sato
Masao Hashimoto
Kenji Watanabe
Yoichi Mizukami
Jun Muto
author_sort Eiko Uno
collection DOAJ
description Hyaluronan (HA) provides moisturizing benefits and exhibits unique biological activities based on its molecular weight. While the anti-inflammatory effects of high-molecular-weight HA have been well studied, the impact of hyaluronan tetrasaccharide (HA4), an ultralow-molecular-weight HA, on the skin immune system is not fully understood. Thus, we investigated how HA4 affects the differentiation of M1 macrophages, which increase during photoaging. As a result, we added HA4 during the M1 macrophage differentiation phase and conducted a gene expression analysis. HA4 partially decreased the transition from M0 to M1 macrophages and reduced the expression of proinflammatory cytokines like IL-6. However, the M2 marker IL-1ra increased, while IL-10 levels remained constant, suggesting that HA4 does not fully polarize macrophages toward the M2 phenotype. Normal human dermal fibroblasts (NHDF) were treated with an M1 macrophage-conditioned medium (M1-CM) and a modified version containing HA4 (M1+HA4-CM). The M1+HA4-CM notably decreased the expression of IL-6 and IL-8, along with the collagen-degrading enzyme MMP1. Collagen synthesis assays showed that HA4 helped restore collagen fiber formation. Moreover, RNA-seq analysis of NHDF treated with the conditioned medium confirmed that M1+HA4-CM amplified the expression of genes related to collagen production while decreasing collagen-degrading enzyme gene expression. Neutralization assays employing a TLR4 antibody suggested that decreasing IL-6 in NHDF by HA4 may be independent of the TLR4 signaling pathway. HA4 is vital in partially suppressing M1 macrophage differentiation and the release of inflammatory factors, as well as regulating collagen remodeling in NHDF. These findings indicate that HA4 holds promise as a molecule for mitigating inflammation-induced collagen degradation by modulating macrophage activity in photoaged skin.
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spelling doaj-art-cc1fa76a23fe436598dd14701b1bd88d2025-08-20T02:03:19ZengFrontiers Media S.A.Frontiers in Immunology1664-32242025-06-011610.3389/fimmu.2025.15927511592751Targeting inflammatory macrophages with hyaluronan tetrasaccharide: effects on fibroblast collagen degradation and synthesisEiko Uno0Florence Kim1Mihoko Yoshino2Yasunari Sato3Masao Hashimoto4Kenji Watanabe5Yoichi Mizukami6Jun Muto7Fundamental Technology Research Division, ROHTO Pharmaceutical Co., Ltd., Kizugawa, Kyoto, JapanFundamental Technology Research Division, ROHTO Pharmaceutical Co., Ltd., Kizugawa, Kyoto, JapanFundamental Technology Research Division, ROHTO Pharmaceutical Co., Ltd., Kizugawa, Kyoto, JapanFundamental Technology Research Division, ROHTO Pharmaceutical Co., Ltd., Kizugawa, Kyoto, JapanFundamental Technology Research Division, ROHTO Pharmaceutical Co., Ltd., Kizugawa, Kyoto, JapanInstitute of Gene Research, Yamaguchi University Science Research Center, Yamaguchi, JapanInstitute of Gene Research, Yamaguchi University Science Research Center, Yamaguchi, JapanDepartment of Dermatology, Ehime University Graduate School of Medicine, Toon, Ehime, JapanHyaluronan (HA) provides moisturizing benefits and exhibits unique biological activities based on its molecular weight. While the anti-inflammatory effects of high-molecular-weight HA have been well studied, the impact of hyaluronan tetrasaccharide (HA4), an ultralow-molecular-weight HA, on the skin immune system is not fully understood. Thus, we investigated how HA4 affects the differentiation of M1 macrophages, which increase during photoaging. As a result, we added HA4 during the M1 macrophage differentiation phase and conducted a gene expression analysis. HA4 partially decreased the transition from M0 to M1 macrophages and reduced the expression of proinflammatory cytokines like IL-6. However, the M2 marker IL-1ra increased, while IL-10 levels remained constant, suggesting that HA4 does not fully polarize macrophages toward the M2 phenotype. Normal human dermal fibroblasts (NHDF) were treated with an M1 macrophage-conditioned medium (M1-CM) and a modified version containing HA4 (M1+HA4-CM). The M1+HA4-CM notably decreased the expression of IL-6 and IL-8, along with the collagen-degrading enzyme MMP1. Collagen synthesis assays showed that HA4 helped restore collagen fiber formation. Moreover, RNA-seq analysis of NHDF treated with the conditioned medium confirmed that M1+HA4-CM amplified the expression of genes related to collagen production while decreasing collagen-degrading enzyme gene expression. Neutralization assays employing a TLR4 antibody suggested that decreasing IL-6 in NHDF by HA4 may be independent of the TLR4 signaling pathway. HA4 is vital in partially suppressing M1 macrophage differentiation and the release of inflammatory factors, as well as regulating collagen remodeling in NHDF. These findings indicate that HA4 holds promise as a molecule for mitigating inflammation-induced collagen degradation by modulating macrophage activity in photoaged skin.https://www.frontiersin.org/articles/10.3389/fimmu.2025.1592751/fullhyaluronan tetrasaccharide (HA4)macrophagesfibroblastsdermisphotoaging
spellingShingle Eiko Uno
Florence Kim
Mihoko Yoshino
Yasunari Sato
Masao Hashimoto
Kenji Watanabe
Yoichi Mizukami
Jun Muto
Targeting inflammatory macrophages with hyaluronan tetrasaccharide: effects on fibroblast collagen degradation and synthesis
Frontiers in Immunology
hyaluronan tetrasaccharide (HA4)
macrophages
fibroblasts
dermis
photoaging
title Targeting inflammatory macrophages with hyaluronan tetrasaccharide: effects on fibroblast collagen degradation and synthesis
title_full Targeting inflammatory macrophages with hyaluronan tetrasaccharide: effects on fibroblast collagen degradation and synthesis
title_fullStr Targeting inflammatory macrophages with hyaluronan tetrasaccharide: effects on fibroblast collagen degradation and synthesis
title_full_unstemmed Targeting inflammatory macrophages with hyaluronan tetrasaccharide: effects on fibroblast collagen degradation and synthesis
title_short Targeting inflammatory macrophages with hyaluronan tetrasaccharide: effects on fibroblast collagen degradation and synthesis
title_sort targeting inflammatory macrophages with hyaluronan tetrasaccharide effects on fibroblast collagen degradation and synthesis
topic hyaluronan tetrasaccharide (HA4)
macrophages
fibroblasts
dermis
photoaging
url https://www.frontiersin.org/articles/10.3389/fimmu.2025.1592751/full
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