Highlights•

uLMW-HA decreased UVB-induced IL-6 secretion.

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uLMW-HA downregulated Calprotectin-induced TRAF6 expression.

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CD44v are involved in HA-regulated anti-inflammatory activity.

AbstractBackground

The biological functions of Hyaluronic acid are related to its molecular weight and binding to its receptor, Toll-like receptor4 (TLR4) or CD44. Recent studies have shown that low-molecular-weight Hyaluronic acid (LMW-HA) exhibits proinflammatory effects, while high-molecular-weight Hyaluronic acid (HMW-HA) functions as an anti-inflammatory factor. UVB-induced epidermal inflammation is mainly mediated by endogenous molecules, such as damage-associated molecular patterns (DAMPs), that cause severe skin damage by activating TLR signaling pathways.

Objective

Since both LMW- and HMW-HA have inhibitory functions on TLR-mediated macrophage inflammation, HA is assumed to suppress UVB-induced DAMP-mediated inflammation in the skin. In this study, both Ultra- low-molecular-weight Hyaluronic acid (uLMW-HA) and HMW-HA were found to inhibit UVB-induced keratinocyte inflammation.

Methods

HaCaT cells were treated with medium containing Hyaluronic acid at the appropriate concentration after 15 mJ/cm2 irradiation. Secreted protein levels were determined with ELISA kits. Expression levels of proteins downstream of TLR4 were detected by Simple Western system.

Results

By competitively binding to TLR4, uLMW-HA downregulated Calprotectin-induced TRAF6 expression, which might be the direct process by which uLMW-HA decreased UVB-induced IL-6 secretion. Reduced　CD44 variant (CD44v) expression in keratinocytes attenuated the inhibitory effect of both uLMW-HA and HMW-HA on UVB-induced inflammation, which indicated the involvement of CD44v in HA-regulated anti-inflammatory activity.

Conclusion

Overall, this research indicates that Hyaluronic acid is more than a moisturizer; it is also a biologically effective material that can prevent the excessive skin inflammation caused in daily life, especially in the late stages after sunburn.

Abbreviations: Has (Hyaluronic acid), ECM (extracellular matrix), LMW-HA (low-molecular-weight Hyaluronic acid), HMW-HA (high-molecular-weight Hyaluronic acid), TLR (toll-like receptor), LPS (lipopolysaccharide), MyD (myeloid differentiation factor), IL (interleukin), IFNs (type I interferons), ROS (reactive oxygen species), DAMPs (damage-associated molecular patterns), FBS (fetal bovine serum), uLMW-HA (ultra- low-molecular-weight Hyaluronic acid), WES (Simple Western), CD44v (CD44 variant), CD44s (CD44 standard), NHEK (normal human epidermal keratinocyte), IPA (Ingenuity pathway analysis)Keywords1. IntroductionHyaluronic acid (HA), a natural linear polysaccharide in the extracellular matrix (ECM), is synthesized by fibroblasts, keratinocytes and endothelial cells in the cutaneous region to regulate several biological processes [[1]Hyaluronic acid in dermatology.]. The molecular size of HA ranges from 0.8 kDa to 3000 kDa, with the size determined by the number of repeating disaccharide units.One of the most well-known functions of HA is moisturization, owing to its good ability to trap water [[2]Witting M. Boreham A. Brodwolf R. Vávrová K. Alexiev U. Friess W. Hedtrich S. Interactions of hyaluronic acid with the skin and implications for the dermal delivery of biomacromolecules.]. HA is also well known to reduce the appearance of wrinkles and accelerate wound healing. In addition to these functions, HA-based formulations have shown remarkable efficacy in treating a wide range of inflammatory skin diseases [3Hong L. Shen M. Fang J. Wang Y. Bao Z. Bu S. Zhu Y. Hyaluronic acid (HA)-based hydrogels for full-thickness wound repairing and skin regeneration., 4Litwiniuk M. Krejner A. Speyrer M.S. Gauto A.R. Grzela T. Hyaluronic acid in inflammation and tissue regeneration., 5Chen L.H. Xue J.F. Zheng Z.Y. Shuhaidi M. Thu H.E. Hussain Z. Hyaluronic acid, an efficient biomacromolecule for treatment of inflammatory skin and joint diseases: a review of recent developments and critical appraisal of preclinical and clinical investigations.]. Studies have also indicated that physiological responses mediated by HA usually correlate with immune functions causing tissue inflammation. Some papers suggest that low-molecular-weight HA (LMW-HA) can induce inflammatory reactions, while high-molecular-weight HA (HMW-HA) functions as an anti-inflammatory factor [[6]Muto J. Sayama K. Gallo R.L. Kimata K. Emerging evidence for the essential role of hyaluronan in cutaneous biology.]. The different signal activation abilities of HAs are based on the diverse molecular sizes, which are related to the different receptors utilized.Toll-like receptor (TLR) 4, an innate receptor of bacterial lipopolysaccharide (LPS), is also an important receptor of LMW-HA [[7]Campo G.M. Avenoso A. Campo S. D’Ascola A. Nastasi G. Calatroni A. Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes.]. TLR4 activates NF-κB via two major pathways: a myeloid differentiation factor (MyD) 88-dependent pathway that leads to NF-κB nuclear translocation to induce the expression of proinflammatory cytokines, such as interleukin (IL) 6, and a MyD88-independent pathway that acts via type I interferons (IFNs) to upregulate IFN-induced proinflammatory gene expression [[8]Lu Y.C. Yeh W.C. Ohashi P.S. LPS/TLR4 signal transduction pathway.]. LMW-HA usually regulates cell biological functions by interacting with the membrane receptor TLR4, while the glycoprotein CD44 is the main binding receptor of HMW-HA. The CD44-mediated pathway leads to normal keratinocyte differentiation and cell survival. Oligo-HA was shown to inhibit TLR3-dependent inflammation via its interaction with TLR4 [[9]Kim M.Y. Muto J. Gallo R.L. Hyaluronic acid oligosaccharides suppress TLR3-dependent cytokine expression in a TLR4-dependent manner.]. Publications have also revealed that 500-kDa HMW-HA protects macrophages from LPS-induced inflammation via an interaction between HMW-HA/CD44 and LPS/TLR4 signals [[10]Muto J. Yamasaki K. Taylor K.R. Gallo R.L. Engagement of CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS.]. These studies provide strong evidence that the functions of HA are related to its binding receptors (TLR4 and CD44) and their relevant signaling pathways.As a kind of external environment-induced skin inflammation, UVB-induced epidermal inflammation is thought to be initiated by reactive oxygen species (ROS) generated by UVB radiation immediately after UV exposure, which is responsible for NF-κB-regulated cytokine release. Additionally, damage-associated molecular patterns (DAMPs), which are endogenous molecules created upon tissue injury, contribute to UVB-induced skin inflammation [[11]Bernard J.J. Cowing-Zitron C. Nakatsuji T. et al.Ultraviolet radiation damages self noncoding RNA and is detected by TLR3.]. Traditional DAMPs, such as long noncoding RNAs, HMGB1, and Calprotectin, usually cause severe skin damage when they are released from cells. Calprotectin is induced in UV-irradiated keratinocyte [[12]Kerkhoff C. Voss A. Scholzen T.E. Averill M.M. Zänker K.S. Bornfeldt K.E. Novel insights into the role of S100A8/A9 in skin biology.], and it influence cell functions through activating TLR signaling pathways [13Damage-associated molecular patterns in inflammatory diseases., 14Toll-like receptor signaling and its inducible proteins.]. For example, Calprotectin, a stable heterodimer of S100A8 and S100A9, can exacerbate inflammation by inducing the secretion of multiple cytokines in inflammatory cells, such as by activating TLR4 to release IL-6 from cells [[15]Ma L. Sun P. Zhang J.C. Zhang Q. Yao S.L. Proinflammatory effects of S100A8/A9 via TLR4 and RAGE signaling pathways in BV-2 microglial cells.]. Additionally, by involving TLR4 downstream signaling through MAPK and NF-κB, Calprotectin induces IL-6 production in gingival fibroblasts in periodontitis patients [[16]Nishikawa Y. Kajiura Y. Lew J.H. Kido J.I. Nagata T. Naruishi K. Calprotectin induces IL-6 and MCP-1 production via toll-like receptor 4 signaling in human gingival fibroblasts.]. Therefore, Calprotectin plays a critical role in the promotion of TLR4-related cytokine secretion under inflammatory conditions. Since both LMW-HA and HMW-HA have inhibitory effects on TLR-mediated macrophage inflammation, HA is assumed to have a high capacity to suppress TLR4-related keratinocyte inflammation, which also includes UVB-induced Calprotectin-mediated skin inflammation.

Here, we tested whether HAs have anti-inflammatory effects on inflammation induced by UVB exposure. Clarification of the detailed mechanism will provide a better understanding of the biological functions of HA in keratinocytes.

2. Materials and methods2.1 Cell cultureThe human keratinocyte cell line HaCaT was obtained from DKFZ (Heidelberg, Germany) [[17]Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.]. HaCaT cells were grown in Dulbecco’s modified Eagle’s medium containing 10 % fetal bovine serum (FBS), penicillin (100 IU/mL), and streptomycin (100 µg/mL). The cells were maintained in an incubator containing 5 % CO2 at 37 °C.

Normal Human Epidermal Keratinocyte (NHEK) was　obtained from KURABO　(Osaka, Japan). NHEK cells were grown in HuMedia-KG2 (KURABO) supplemented with the HuMedia-KG supplement kit (KURABO). The cells were maintained in an incubator containing 5 % CO2 at 37 °C.

2.2 Reagents

Ultra-LMW (uLMW)-HA (0.8 kDa), which is a tetra saccharide, was synthesized and purified by ROHTO Pharmaceutical Co., Ltd. HMW-HA (1200 kDa) was purchased from Iwaki Seiyaku Co., Ltd. (Y-944). Both HAs are LPS-free specification. Recombinant Calprotectin used to initiate IL-6 secretion was purchased from R&D Systems (8226-S8), and LPS was obtained from Sigma (L2630).

2.3 UVB irradiation

HaCaT cells (5 × 105 cells/well) were seeded in 6-well plates and cultured overnight. Cells at 80 % confluence were washed twice with 2 mL PBS before irradiation. The UVB source was a broadband UVB device, DERMARAY-200. The doses of UVB irradiation were determined with a UV radiometer, and the total energy of irradiation was 15 mJ/cm2 or 30 mJ/cm2. Secreted IL-6, IL-8, IL-1β, TNFα, and Calprotectin levels were determined with ELISA kits as described below. In HA treatment experiments, cells were treated with medium containing HA at the appropriate concentration after 15 mJ/cm2 irradiation.

2.4 Calprotectin stimulation

HaCaT cells (5 × 104 cells/well) were seeded in 24-well plates and cultured for 24 h. Cells at almost 80 % confluency were treated with Calprotectin only or Calprotectin with different HAs at the appropriate concentrations. Total protein was collected 4 h after stimulation, and MyD88 and TRAF6 expression was evaluated by Simple Western system (WES). Supernatants collected 72 h after stimulation were evaluated with an IL-6 ELISA kit as described below.

2.5 ELISA kits

The concentrations of IL-6, IL-8, TNFα, IL-1β and Calprotectin in HaCaT cell supernatants were evaluated using ELISA kits obtained from R&D Systems (IL-6, D6050; IL-8, DY208; IL-1β, DLB50; TNFα, DTGA00; Calprotectin, DS8900) according to the manufacturer’s instructions.

2.6 Western blot analysis

Total protein was extracted from treated and untreated HaCaT cells using M-PER protein extraction reagent (Thermo Fisher Scientific, 78 501) supplemented with protease and phosphatase inhibitors. MyD88, TRAF6, and Tubulin expression levels were determined by WES. Anti-MyD88 and anti-TRAF6 antibodies were purchased from Cell Signaling Technology (D80F5 and D21G3). An anti-Tubulin antibody was obtained from Abcam (ab7291).

2.7 CD44v-knockdown experiments

SMARTpool-siRNA targeting human CD44v was designed and synthesized by Horizon. CD44v expression was downregulated using CD44v-specific siRNA, and this siRNA or negative control siRNA was transfected into cells for 24 h using Lipofectamine RNAiMAX Reagent (Invitrogen, 13 778) according to the manufacturer’s instructions. Then, the cells were used for the experiments 6 days after lipofectamine treatment. The expression levels of CD44v and TLR4 on day 6 were tested using WES. An anti-CD44 antibody was purchased from NOVUS Biologicals (NBP1–47 386), and an anti-TLR4 antibody was obtained from Santa Cruz Biotechnology (sc-293 072).

2.8 qPCR analysis

Total RNA was extracted from the HaCaT cells with the RNeasy 542 Mini Kit (Qiagen, Hilden, Germany). The procedure of the complementary DNA (cDNA) libraries was carried out with ReverTra Ace® qPCR RT Master Mix (Toyobo, Japan). qPCR analysis was conducted using QuantoStudio 7 (Thermo Fisher Scientific, USA).

2.9 Statistical analysis

Data are presented as the mean ± SD. Student’s t-test was used to compare the control group with the Calprotectin-stimulated group. Dunnett's test was performed where appropriate to compare the control group with treated groups. Statistical significance was defined as p < 0.05.

2.10 Whole transcriptome analysis with RNA-seq

Total RNA was extracted from the HaCaT cells with the RNeasy 542 Mini Kit (Qiagen, Hilden, Germany), and mRNA was purified with oligo dT beads (NEBNext Poly (A) mRNA magnet Isolation Module, New England Biolabs, NEB, Ipswich, MA). The procedure of the complementary DNA (cDNA) libraries was carried out with NEBNext Ultra II RNA library Prep kit (NEB) and NEBNextplex Oligos for Illumina following a previously described method (Kohno et al., 2020). The index sequences were inserted to the fragments with PCR amplification. The cDNA libraries were added in equal molecular amounts and were sequenced on an Illumina Next-seq DNA sequencer with a 75-bp pair-end cycle sequencing kit (Illumina, San Diego, CA). The detected reads were analyzed by using CLC Genomics Workbench software (ver.8.01, Qiagen). The pathway of the detected genes was analyzed using Ingenuity pathway analysis, called IPA (Qiagen).

3. Results3.1 IL-6 and IL-8 expression is significantly upregulated under UVB irradiationUVB-induced HaCaT cell inflammation was confirmed by measuring the secreted levels of the proinflammatory factors IL-6, IL-8, TNFα and IL-1β (Fig. 1A-D). HaCaT cells were irradiated with UVB at doses of 0, 15, and 30 mJ/cm2 using broadband UVB devices. The levels of all of these proinflammatory factors were significantly increased in a UVB dose-dependent manner. In particular, the release of IL-6 was 12 times higher at 15 mJ/cm2 and 37 times higher at 30 mJ/cm2 UVB than at 0 mJ/cm2. The lower dose of 15 mJ/cm2 was chosen to simulate weaker skin inflammation conditions in later experiments because of the increased proinflammatory cytokine release and the cell survival rate greater than 70 % (data not shown).

Fig. 1Effects of UVB on secretion of inflammatory cytokines IL-6, IL-8, TNFα, and IL-1β in HaCaT cells and NHEK cells. HaCaT cells were irradiated by UVB at doses of 0, 15 and 30 mJ/cm2. Secretions of (A)IL-6, (B)IL-8, (C)TNFα, (D)IL-1β were determined by ELISA. Increased secretion of IL-6, IL-8, TNFα and IL-1β was observed with UVB radiation strength. NHEK cells were irradiated by UVB at doses of 0, 15 and 30 mJ/cm2. Secretions of (E)IL-6, (F)IL-8 and (G)IL-1β were determined by ELISA. Increased secretion of IL-6, IL-8 and IL-1β was observed with UVB radiation strength.　TNFα was not detected at UVB　doses of 0 and 15 mJ/s. Error bars reveal the means±standard deviation in n = 6 tests. **P < 0.01 as compared to unirradiated cells.

Secreted levels of IL-6, IL-8 and IL-1βwere measured by ELISA (Fig. 1E-G). NHEK cells were irradiated with UVB at doses of 0, 15, and 30 mJ/cm2 using broadband UVB devices. The levels of these proinflammatory factors were significantly increased in a UVB dose-dependent manner. TNFα was not detected. The UVB-induced NHEK cell inflammation is consistent with the UVB-induced HaCaT cell inflammation.3.2 HA inhibits UVB-induced IL-6 release from HaCaT cellsTo clarify the effects of HA on UVB-induced keratinocyte inflammation, HA treatment was performed after UVB irradiation. Since the inflammatory cytokines IL-6, IL-8, TNFα, and IL-1β were released from UVB-irradiated HaCaT cells, their secreted levels were tested by ELISA after treatment with HA. uLMW-HA and HMW-HA were found to have inhibitory effects on IL-6 secretion (Fig. 2A) but were not able to suppress IL-8, TNFα, or IL-1β secretion (Fig. 2B-D), which indicated that the regulation of IL-6 secretion might be different from that of the other 3 cytokines in the context of treatment with HA. Inhibitory effect of HA on UVB-induced keratinocyte inflammation was also examined with NHEK. Secreted levels of IL-6, IL-8 and IL-1β from UVB-irradiated NHEK after HA treatment were tested by ELISA (Fig. 2E-G). The results showed that uLMW- and HMW-HA suppressed IL-6 and IL-8 secretion from NHEK. Since IL-6 secretion was reduced only 15–20 % in the HA-treated HaCaT cells compared with the non-HA-treated group, HA was shown to have a limited ability to relieve UVB-induced skin inflammation.

Fig. 2Effects of HA on IL-6, IL-8, TNFα, and IL-1β production in UVB-irradiated HaCaT cells and NHEK cells. HaCaT cells were irradiated with 0 or 15 mJ/cm2 of UVB, then treated with various concentrations (0.1 or 1 mg/mL) of uLMW-HA or HMW-HA for 72 h. (A)IL-6, (B)IL-8, (C)TNFα, and (D)IL-1β production were determined by ELISA. Although secretion of IL-8, TNFα and IL-1β was not suppressed significantly by both uLMW-HA and HMW-HA, IL-6 was significantly suppressed by 0.1 or 1 mg/mL uLMW-HA or 1 mg/mL HMW-HA. NHEK cells were irradiated with 0 or 15 mJ/cm2 of UVB, then treated with various concentrations (0.1 or 1 mg/mL) of uLMW-HA or HMW-HA for 72 h. (E)IL-6, (F)IL-8 and (G)IL-1β production were determined by ELISA. IL-6, IL-8 and IL-1β was significantly suppressed by 1 mg/mL uLMW-HA or 1 mg/mL HMW-HA. The data represent the means ± SD of triplicate experiments. *p < 0.05, **p < 0.01 compared to UVB expose alone.

To confirm uLMW-HA or HMW-HA inhibiting UV-induced inflammation, whole transcriptome analysis was conducted. The volcano plot shows the significantly upregulated (red) and downregulated (green) genes of more than 20 % with less than 0.05 p-values by UVB radiation. (Supplementary Fig S1). We analyzed network of these genes with Ingenuity Pathway analysis using information collected from databases on protein interactions. IL-6 was detected as an upstream factor of HA suppressing UV-radiation effects on HaCaT cells (Supplementary Fig S2–5). Canonical pathway analysis also shows uLMW-HA downregulated NF-κB, which is located downstream of TLR4 signaling pathway (Supplementary Fig S46), although HMW-HA did not show downregulation of NF-κB signaling pathway.3.3 HA attenuates Calprotectin-induced inflammation by decreasing IL-6 releaseIn addition to the reactions influenced by UVB-initiated ROS, DAMPs created upon UVB damage also induce skin inflammation in the late stages after sunburn. Continuous stimulation by DAMPs also exacerbates the inflammatory condition in the skin, which hinders recovery. DAMPs are molecules that usually stimulate the innate immune system by binding to TLRs. Based on current studies, it is assumed that HA may suppress TLR4-regulated inflammation, so we investigated the release of TLR4-binding DAMPs. Besides the DAMPs we checked (data not shown), the secretion of one type of DAMP, Calprotectin, was significantly increased with UVB exposure. The secreted level changed by more than 1.6-fold after 15 mJ/cm2 UVB irradiation (Fig. 3A). To further clarify whether HA specifically targets Calprotectin/TLR4 activation, the influence of HA on Calprotectin-induced IL-6 release was tested by ELISA. A high concentration (1 mg/mL) of uLMW-HA or HMW-HA reduced the total amount of IL-6 in the supernatant to the level of that in the non-Calprotectin-stimulated group. The inhibitory effects of uLMW-HA and HMW-HA on IL-6 secretion provided strong evidence that HA can block TLR4 activation effectively (Fig. 3B, C).

Fig. 3Effects of HA on Calprotectin-induced IL-6 secretion. (A)Secretion of Calprotectin from HaCaT cells was evaluated by ELISA under irradiation of 0, 15, and 30 mJ/cm2 of UVB. **p < 0.01 compared to non-UVB irradiation. Effects of (B)uLMW-HA or (C)HMW-HA on calprotectin-induced IL-6 secretion were determined by ELISA. It was shown that secretion was significantly suppressed by 1 mg/mL uLMW-HA or 1 mg/mL HMW0-HA. ##p < 0.01 compared to non-Calprotectin treatment. *p < 0.05, **p < 0.01 compared to non-HA treatment. The data represent the means ± SD of triplicate experiments.

3.4 uLMW-HA downregulates while HMW-HA upregulates TRAF6 expression in HaCaT cellsTo further confirm the activation conditions of the Calprotectin/TLR4 cascade, western blotting was utilized to evaluate protein expression levels. The level of the protein MyD88, which is recruited to the TLR4 region immediately after ligand stimulation, did not vary with uLMW-HA treatment (Fig. 4A). uLMW-HA treatment groups exhibited a reduced amount of total TRAF6 protein after Calprotectin stimulation compared to the non-HA-treated group (Fig. 4B). These results indicate that uLMW-HA may reduce Calprotectin-induced IL-6 production by decreasing the total protein level of TRAF6, which contributes to the inhibition of UVB-related keratinocyte inflammation. In contrast, increased expression levels of TRAF6 were found with HMW-HA treatment after Calprotectin stimulation (Fig. 4D), which suggested the involvement of other signaling pathway interactions.

Fig. 4Expression levels of TLR4 downstream protein MyD88 and TRAF6. Protein expression levels of MyD88, TRAF6 and tubulin were determined by Simple Western (WES). Quantitively analysis were shown in bar graph relative to control group. (A) MyD88 and (B)TRAF6 showed the expressions under uLMW-HA treatment. (C) MyD88 and (D)TRAF6 presented the expressions under HMW-HA treatment.

3.5 Reduced CD44v expression does not influence the anti-inflammatory effects of uLMW-HA but does weaken the inhibitory effect of HMW-HA on UVB-stimulated inflammationTo clarify the involvement of CD44 in the anti-inflammatory effect of HMW-HA, CD44 expression was down-regulated in HaCaT cells. Since research showed that HaCaT cells express more CD44 variant (CD44v) compared to CD44 standard (CD44s) [[18]Montero-Montero L. Renart J. Ramírez A. Ramos C. Shamhood M. Jarcovsky R. Quintanilla M. Martín-Villar E. Interplay between Podoplanin, CD44s and CD44v in squamous carcinoma cells.], siRNA targeting CD44v was utilized to reduce CD44v expression. Western blotting was utilized to confirm the knockdown efficiency. The bar graph showing quantified data indicates that the CD44v-knockdown expression level was 60 % of the normal level, while the CD44s form did not exhibit a change in expression (Fig. 5A). The gene expression analysis also shows suppression of CD44v expression (Supplementary Fig. S7). siRNA treatment did not influence TLR4 expression. In CD44v-downregulated cells, the effects of HA on UVB-induced IL-6 secretion were tested. A low concentration of uLMW-HA produced weaker inhibitory effects on IL-6 secretion in CD44v down-regulated cells than in control cells, but decreased CD44v expression did not influence the effects of a high concentration of uLMW-HA. On the other hand, CD44v reduction in HaCaT cells prevented the continuous inhibitory effect of HMW-HA on inflammation stimulated by UVB (Fig. 5C), However, a high concentration of HMW-HA maintained the function of reducing IL-6 release. The results indicate that CD44v influences both the function of low concentration uLMW-HA and HMW-HA.

Fig. 5Effect of HA on UVB-irradiated CD44 knockdown HaCaT cells. （A)Knockdown efficacy of CD44 was checked by WES. It was also showen the quantitative data of CD44s, CD44v and TLR4 expression in KD HaCaT cells. CD44v　was successfully knockdowned and the expression of CD44s and TLR4 was not affected.　(BEffects of (B)uLMW-HA or (C)HMW-HA on calprotectin-induced IL-6 secretion were determined by ELISA. *p < 0.05, * *p < 0.01 compared to non-HA treatment. The data represent the means ± SD of triplicate experiments.

4. DiscussionIt is known that acute inflammation after UVB irradiation is always accompanied by oxidative stress induced by rapid ROS accumulation [[19]Subedi L. Lee T.H. Wahedi H.M. Baek S.H. Kim S.Y. Resveratrol-enriched rice attenuates UVB-ROS-induced skin aging via downregulation of inflammatory cascades [published correction appears in.]. The important process during inflammation is the nuclear translocation of the transcription factor NF-κB, which activates the expression of many genes involved in inflammation. The canonical pathway of NF-κB activation is regulated by several stimuli, such as TLR agonists, that can activate TRAF6 and lead to the activation of the IKK complex downstream, resulting in the phosphorylation of IκBα. Additionally, UVB-induced ROS can also initiate NF-κB activation independently of the IKK complex by directly phosphorylating other protein residues in IκBα [[20]NF-κB signaling pathway and free radical impact.]. Although uLMW-HA attenuates TLR signaling via TRAF6, the downstream cascade can still be activated by ROS via IKK-independent IκBα activation. uLMW-HA is thought to weaken only the inflammatory effects initiated by TLR4. Moreover, considering the speed of ROS generation, TLR-initiated NF-κB nuclear translocation will be comparably late because it is mostly activated by proinflammatory cytokines through an autocrine or paracrine pathway. Therefore, the results in Fig. 2A indicate that inflammation suppression by HA may not be directly linked to ROS-related acute reactions. Although the inhibitory effect of HA on UVB-stimulated inflammation is very weak, the blockade of TLR4 signaling by HA may attenuate the late keratinocyte inflammation regulated by DAMPs. Moreover, as shown in Fig. 3B, the effect of HA on IL-6 release from keratinocytes stimulated with Calprotectin was not as obvious; IL-6 release was increased less than 20 % compared to control release. Although Calprotectin was shown to play an important role in TLR4-regulated inflammation, it seemed to play only a small part in UVB-induced inflammation. This also explains why HA could block only 20 % of IL-6 secretion after UVB exposure.

Although the efficacy of IL-6 secretion blockade was not obvious and the release of IL-8, TNFα, and IL-1β was not inhibited by HA, the application of HA in skin diseases is still desirable. The reason is that the increased level of secreted IL-6 has been indicated to be associated with a number of skin pathologies. Highly expressed IL-6 in psoriatic skin stimulates abnormal keratinocyte proliferation. IL-6 inhibition by HA is helpful in maintaining skin homeostasis.

It is thought that both uLMW-HA and HMW-HA exhibit anti-inflammatory functions in the context of UVB-induced inflammation by inhibiting DAMP-mediated TLR4 pathway activation. The binding of uLMW-HA for TLR4 may allow uLMW-HA to competitively bind to TLR4 to block the binding of other TLR4 ligands, such as calprotectin, which leads to the downregulation of TRAF6 activity downstream of TLR4 signaling. As a result, IL-6 secretion from keratinocytes is reduced. Interestingly, stimulation with Calprotectin only slightly downregulated TRAF6 expression. In other studies, stimulation with TLR ligands, such as LPS, led to the downregulation of endogenous TRAF6 expression depending on the negative regulatory effect of IRAK1 on TLR signaling [[21]IRAK-1-mediated negative regulation of Toll-like receptor signaling through proteasome-dependent downregulation of TRAF6.]. This may also explain why treatment with uLMW-HA after Calprotectin stimulation dramatically downregulated TRAF6 expression levels.As shown in Fig. 4D, HMW-HA suppressed the expression of TRAF6 without Calprotectin treatment. The likely reason might be the signal masking function of HMW-HA. Binding of HMW-HA to CD44v interrupts the binding of other ligands to their receptors, which attenuates the activity of TLR4, IL1R or TNFR that leads to TRAF6 downregulation. However, HMW-HA upregulated TRAF6 expression in the context of Calprotectin stimulation. The detailed mechanism by which HMW-HA/CD44v cascade activation inhibits calprotectin/TLR4 signaling needs further study. It is possible that the binding of HMW-HA to CD44v inhibits signal transduction proteins downstream of TRAF6 or regulates the activation of TRAF6. TRAF6 is a protein that controls the fate of the TLR4-dependent inflammatory pathway via modification by ubiquitination. Usually, TRAF6 forms a homodimer and catalyzes lysine 63 (K63)-linked ubiquitination of itself to activate the TAK1 pathway [[22]Wang G. Gao Y. Li L. et al.K63-linked ubiquitination in kinase activation and cancer.]. K63-linked protein ubiquitination does not target proteins for proteasomal degradation but instead serves as a scaffold to connect proteins in signaling pathways. Therefore, the K63-linked ubiquitination of TRAF6 is essential for the activation of TLR4-dependent NF-κB signaling. Although HMW-HA upregulated the total protein level of TRAF6, treatment with HMW-HA may decrease the K63-linked ubiquitination of TRAF6 to inhibit IL-6 release. Moreover, the TRAF6 protein condition is also regulated by the negative regulator A20, which is a protein that regulates TRAF6 enzymatic activity by direct deubiquitylation [23Mabilleau G. Chappard D. Sabokbar A. Role of the A20-TRAF6 axis in lipopolysaccharide-mediated osteoclastogenesis., 24Traf6 and A20 differentially regulate TLR4-induced autophagy by affecting the ubiquitination of Beclin 1.]. By signaling through CD44v, HMW-HA might downregulate the level of the ubiquitinated form of TRAF6 to attenuate TLR4-mediated IL-6 secretion, even though the total protein level was upregulated.Although publications have shown that the inhibitory effects of LMW-HA on TLR4 signaling are independent of CD44 expression [[24]Traf6 and A20 differentially regulate TLR4-induced autophagy by affecting the ubiquitination of Beclin 1.], the results in Fig. 5 indicate that the interaction between TLR4 and CD44v is required to regulate uLMW-HA functions. Moreover, other research groups have reported that CD44 and TLR4 colocalize upon treatment with HA [10Muto J. Yamasaki K. Taylor K.R. Gallo R.L. Engagement of CD44 by hyaluronan suppresses TLR4 signaling and the septic response to LPS., 25