Modulation of wound healing regulators by thermal spring water from La Bourboule

doi:10.21203/rs.3.rs-2889930/v1

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Modulation of wound healing regulators by thermal spring water from La Bourboule

https://doi.org/10.21203/rs.3.rs-2889930/v1

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Thermal spring waters have been a source of treatment for curing or alleviating symptoms of various pathologies. However, despite some supporting literature of the late 19^th century, there has been a gradual decrease in their use as the exact mechanisms remained unknown. The thermal spring water from La Bourboule (BW) in Central France, is still being used for treating skin diseases. To decipher how BW has beneficial effects on wound healing, we cultured HaCaT keratinocytes with increasing percentages of BW and analysed their properties by a scratch assay. Production of cytokines, metallopeptidases, and accumulation of mRNA of genes encoding proteins involved in wound healing processes were also investigated. Our results show that BW significantly increases the wound healing capacities of HaCaT cells by 12% at 24h, paralleled with a significant increase of TGFb1-3 mRNA accumulation (1.3 to 2.2-fold) and TGFb1 production (1.3-fold) at 24h. Conversely, MMPs’ mRNA and/or peptides are reduced. IL6, TNFa and other factors playing in the wound healing processes are modulated at transcript and/or protein levels. Altogether, this study suggests that BW modulates, in vitro, the growth and functioning of keratinocytes through molecular mechanisms, which deserve to be investigated further.

Biological sciences/Biochemistry/Cytokines/Interleukins

Earth and environmental sciences/Hydrology

thermal spring water

keratinocytes

wound healing

inflammation

In 1879, the British Medical Journal reported that, as early as 1540, a specific thermal spring water from La Bourboule (BW) was used for treating human skin diseases (Brandt 1879). La Bourboule is a French commune in a natural park located in the Auvergne-Rhône-Alpes region (N 45°35′21’’, E 2°44′24”) at the foot of Puy de Sancy, an ancient stratovolcano, which has been inactive for about 220,000 years. BW is still being used in balneotherapy. Moreover, its applications have now been extended to respiratory and otorhinolaryngology diseases in addition to cutaneous injuries (Martin et al. 1979; Fauquert and Labbé 1990). Although inflammation modulation has been suspected and “the superior richness in arsenic of the springs of La Bourboule gives its waters a wider range of efficacy in treating skin-affections” (Rabagliati 1880), its effects on the skin physiology has never been investigated at the molecular level. Interestingly, thermal water treatment for upper respiratory tract diseases has shown a significant improvement of the mucociliary clearance time (Keller et al. 2014). This suggests that the application of thermal water can serve as an additional non-pharmacological alternative for healthcare (Keller et al. 2014). Furthermore, Prandelli et al. (Prandelli et al. 2013) reported that a sulphurous thermal water could enhance the release of the anti-inflammatory cytokine IL10 in vitro from human monocytes and in saliva from some patients with chronic upper airway disease. These results represent early evidence that sulphur-based compounds may be useful in treating chronic inflammatory ailments, despite the mechanism remaining unknown. More recently, Aversano et al. reported that a thermal mineral water showed an in-vitro anti-inflammatory activity by down-modulating cyclooxygenase (COX)-2 and matrix metallopeptidase (MMP) 2 proteins (Aversano et al. 2020). Inflammation is an important step of wound healing and begins after injury. During this phase, damaged cells, pathogens, and bacteria are removed from the wounded area. Inflammation is thus an essential step of the wound healing process and is considered as problematic only if prolonged or excessive. Basal keratinocytes from wound edges have also an important role in the epithelialization process (Peplow and Chatterjee 2013). Indeed, these cells migrate without first proliferating, however delivering extracellular matrix (ECM) and integrins to the cell surface by vesicle transport. Moreover, in the early phase of any injury, living cells undergo functional and physical stress and require cholesterol to maintain the integrity of the cell membrane for wound healing (Biswas et al. 2019). Cholesterol has another role in wound healing since the vesicular trafficking described above is tightly modulated by cholesterol-dependent mechanisms (Enrich et al. 2015). Cholesterol also contributes in lamellar body formation for skin barrier repair (Schmitz and Müller 1991). Living cells strongly control their cholesterol content by regulating the expression and/or activity of players orchestrating cholesterol homeostasis, including ABCA1, ABCG1 and IDOL (Xh and Ck 2022).

Because BW is sulphurous, we hypothesized that it may modulate gene expression involved in wound healing and contribute to early reported curative effects (Brandt 1879; Rabagliati 1880). To address this hypothesis, we first characterized BW composition and we further studied the cytotoxicity of BW on the survival of HaCaT keratinocytes, as well as the putative effect of BW on re-epithelialization. Synthesis and release of wound healing players, including cytokines, MMPs and that of cholesterol homeostasis were measured as well. Altogether we show that BW accelerates wound healing in vitro and modulates the synthesis and release of chemokines by keratinocytes.

2.1. Cell culture

HaCaT (kindly provided by Dr. Simon Broad, Centre for Stem Cells & Regenerative Medicine, King's College London, UK) are spontaneously transformed keratinocyte cell line derived from adult human skin (Boukamp et al. 1988). HaCaT cells were regularly and routinely tested for mycoplasma during analysis. Cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Life Technologies®, St Aubin, France), with 100 mg.ml^− 1 penicillin/streptomycin (Life Technologies®) supplemented with 10% fetal bovine serum (FBS, Biowest, Nuallié, France). Cell growth was performed in a humidified incubator containing 5% CO₂ at 37°C. In indicated assays, desteroidated FBS was used to supplement medium. Desteroidated serum was performed with activated charcoal (10mg/ml, Sigma-Aldrich, L’Ile d’Abeau, France) before filtration.

2.2. Thermal spring water analysis and preparation

Thermal spring water was collected from the Choussy-Perriere drilling site (Latitude N 45°35’19.512’’, Longitude E 2°44’13.63244; GPS location 45.5887533,2.73712), currently managed by the municipality of La Bourboule and thermal company called “Grands Thermes de La Bourboule”, from a service pipe directly connected at an emergence point close to the drilling. The tap end was sterilized with a flame and initial flow withdrawn prior to sample collection. Samples for external quality check and characterisation were collected in specific CARSO-LSEHL bottles and analysed by CARSO, a French Health Department accredited laboratory. Analysis began on the collection day and results were available 3 months later. Water quality was assessed before biological effect analysis.

For biological investigation, BW was collected by JMAL and ZGO in sterile dedicated Pyrex® laboratory bottles, cooled at + 2–6°C before cell culture medium preparation, filtration, and conditioning. To prepare control or BW-containing medium, distilled water or BW was used to dissolve DMEM powder as recommended by manufacturer. Mixture was buffered with NaHCO3 (Sigma-Aldrich®), prior to pH adjusting, filtration on 0.2µm filters and addition of penicillin/streptomycin (100 mg.ml^− 1). Conditioned BW containing medium was stored at + 4°C and microbiological and physicochemical stability was checked in comparison to control medium before use. Supplementation with 10% FBS was done extemporaneously.

2.3. Cell counting assay

Cells were seeded and incubated overnight. Control or BW (1 to 10%) containing medium was added the next day. At indicated timing, cells were trypsinized and counted using a LUNA-II® Automated Cell Counter.

2.4. In vitro wound healing assay

Cells were seeded and grown to confluence for 24 hours. Using a sterile micropipette tip, a scratch was created on cell monolayer. Detached cells were then removed with phosphate buffered saline 1X (Life Technologies®). Cells were next grown in the previously indicated conditions. Cell monolayers were photographed (100× magnification) at 24h and 48h following scratching. Wound areas were quantified with ImageJ free software and wound healing was reported as a proportion of remaining area over initial scratch area.

2.5. RNA purification and real-time PCR

Growing cell layer was primarily washed with phosphate buffered saline 1X before lysing for RNA extraction. Total RNA was extracted using TRIzol reagent (Life Technologies®) and cDNA was synthesized with 200 U of Moloney murine leukemia virus-reverse transcriptase (Promega, Charbonnières-les-Bains, France), 5 pmol of random primers (C1181, Promega), 40 U RNAsin (Promega), and 2.5 mM deoxynucleotide triphosphate. Real-time PCR measurement of cDNA was performed using SYBR green dye (Master mix Plus for SYBR Assay, Eurogentec, Angers, France) to measure duplex DNA formation with Eppendorf-Realplex system. Standard curves were generated with pool of cDNA from all samples. Results were analyzed using ΔΔct method. Gene encoding 18S rRNA was used as reference. Primer sequences are given on supplementary table S2.

2.6. Cytokine and metallopeptidase Luminex assay

Cells were grown in a 6-well plate with 2 ml of growth medium per well of. At harvesting time, growth medium was collected for analysis. Cytokines and MMPs were detected using Luminex technology and Human MMP panel 2 (HMMP2MAG-55K-03, Merck-Millipore, Molsheim, France), Human Cytokine panel (HCYTOMAG-60K-02, Merck-Millipore, Molsheim, France) and TGFβ single plex kit (TGFBMAG-64K-01, Merck-Millipore, Molsheim, France), according to manufacturer instructions. Each experiment has been performed in triplicate, supernatants were collected in triplicate, and quantifications of each sample were performed in duplicate. Concentration was obtained by interpolating FI to a dilution standard curve over at least 7 dilution points supplied with kit and calculated using a 5PL curve by Bio-Plex Manager 5.0 software (Bio-Rad). Results were expressed in pg/mL that equal nanograms per liter (ng/L). Standardization curves and limit of detection are available on request.

2.7. Statistics

Values are expressed as means ± standard error mean values. Statistical comparisons, as indicated in figure captions, were performed using a two-tailed Student’s t test or analysis of variance. Significance was accepted for a p value < 0.05.

3.1. Physical and chemical characteristics of the Thermal spring water from La Bourboule

An analysis of BW is being carried out every year by way of documenting its quality in order to certify its safety for use in balneotherapy. The results of the analyses performed at the period we collected water for this study are shown in the supplementary table S1. It depicts its quality regarding the content of certain natural chemicals and estimates the level of risked pollutants and water-related living pathogens. Besides natural chemicals usually found in spring water from volcanic grounds, the analysis did not detect preoccupying levels of industrial or agricultural pollutants.

3.2. Effect of the thermal spring water of La Bourboule on HaCaT cell growth

HaCaT cells were grown with indicated concentrations of BW. At days 1 and 2, living cells were verified by Trypan blue staining and growth was checked by automated counting. As shown in Fig. 1A, in control condition (0% BW), cell number increased 4.8-fold after 24h. Media with BW 1 to 4% did not show any significant effect. Conversely, a significant cell growth inhibition is observed with BW 10% starting at day 1. Media with BW 5% or 6% significantly reduced cells number after 48h (43% and 33% of inhibition, respectively). For further investigations, we chose concentrations lower than or equal to 5% BW. Besides, 4% BW did not induce cell death at 24h and 48 h of growth (Supplementary figure S1).

A preliminary step of wound healing is keratinocyte migration prior to proliferating. To test the influence of BW on keratinocytes repairing capacity, a scratch wound healing assay was performed (Fig. 1B-E). As shown in Fig. 1F, the remaining wound gap is significantly decreased by 12% with 4% BW compared to the control. A proliferative effect of BW was excluded by cell count (Fig. 1A) and MTT (Supplementary figure S1) assay. We concluded that a significant improvement in scratch repair was due to an increased cell migration.

3.3. Thermal spring water of La Bourboule upregulates the TGFβ pathway and downregulates the MMP release

Early studies revealed that TGFβ1 clearly enhances in vivo wound healing (Sporn et al. 1983). It participates in the main steps of this complex process by promoting inflammation and cell migration, proliferation of fibroblasts for ECM formation and tissue construction (Morikawa et al. 2016), and thus repressing expression of MMPs (Santibáñez et al. 2002). To describe BW effect on TGFβ pathway activation, keratinocytes were grown with BW for extraction and quantification of mRNA encoding TGFβ1, 2, 3 and their receptors TGFBR, as well as evaluation of secreted TGFβ1 peptides. In Fig. 2A, mRNA levels of TGFβ family cytokines significantly increase with the amount of BW in medium after 24 h. Conversely, levels of their receptors did not change (Supplementary figure S2). Released TGFβ1 increases following TGFB1 mRNA accumulation (Fig. 2B). Furthermore, paralleling TGFβ induction, MMPs were downregulated at the mRNA (Fig. 2A) and protein (Fig. 2B) levels. Values obtained at 24h and 48h are in accordance (Supplementary figure S3).

3.4. Thermal spring water of La Bourboule increases the accumulation of IL6 and TNFα mRNA in keratinocytes and release of corresponding cytokines

Interleukin 6 (IL6) and TNFα are cytokines involved in the regulation of the immune system, inflammation and wound healing. The S-based compounds NaSH and sulphurous thermal water have been reported to influence cytokine secretion including IL6 and TNFα in inflammatory and basal conditions (Prandelli et al. 2013). As BW is also a sulphurous thermal water, we investigated the production of such cytokines by the keratinocytes in response to BW at 24h (Supplementary figure S4) and 48h (Fig. 3). Interestingly, we found that increased concentrations of BW positively correlated with subsequent increases in IL6 and TNFα at the mRNA (Fig. 3A) and protein (Fig. 3B) level.

3.5. Thermal spring water of La Bourboule modulates the accumulation of mRNA involved in cell cholesterol trafficking

Because production and cell surface delivery of ECM components are modulated by a cholesterol dependent trafficking mechanism (Enrich et al. 2015), we investigated the modulation, by BW, of genes involved in cholesterol homeostasis regulation. Messenger RNA encoding ATP-binding cassettes (ABC) A1 and G1 membrane transporters, which control cholesterol efflux, are significantly down regulated with BW4% by 45 and 35%, respectively (Fig. 4). In a same line of evidence, inducible degrader of the low-density lipoprotein receptor (IDOL), an E3 ubiquitin ligase that triggers the degradation of this receptor, is also downregulated with BW4% or 5%. Conversely, no effect of BW was observed on accumulation of mRNA of E-cadherin, the epithelial marker encoding the CDH1 gene. These data suggest that BW may trigger cholesterol retain in keratinocytes while reducing extracellular cholesterol.

In order to decipher the molecular mechanisms involved in the historically reported effects of thermal spring water from La Bourboule on skin diseases (Brandt 1879; Rabagliati 1880), we studied effects of BW on keratinocytes. Wound healing, MMP secretion and inflammatory cytokine production were investigated.

In vitro wound healing test evidences an accelerating effect of BW (Fig. 1). TGFβ pathways have been previously reported to participate in wound healing by stimulating keratinocyte migration for re-epithelialization and fibroblast activation through epithelial-mesenchymal transition (EMT) activation (Lamouille et al. 2014; Seo et al. 2018). Our data reveal that BW does not induce full activation of EMT due to the absence of loss in epithelial marker CDH1 mRNA accumulation (Fig. 4). In accordance with wound healing enhancement, BW induces the mRNA accumulation of TGFB1, TGFB2 and TGFB3 in HaCaT cells, and increases the release of the cytokine TGFβ1. Since TGFBR mRNA does not decrease, these results suggest a possible autocrine activation (Fig. 2). This is strengthened by the MMP downregulation induced by BW that accompanies the TGFβ upregulation. In accordance, previous studies have reported that sulphurous water could induce the release of TNFα and IL6 by cultured monocytes (Prandelli et al. 2013); however, the precise mechanism has not been identified so far. Contribution of MMPs in wound healing, inflammation and immune system modulation has been clearly evidenced and broadly accepted, even though, using MMP inhibitors in clinical trials is sometimes disappointing. This underlies the necessity to choose selective MMP inhibitors, to target the diseases and to use an efficient dose and the correct time course (Xue et al. 2006; Fields 2019). Interestingly, our observations of increased cell motility being associated with a decrease in MMPs is in accordance with reported animal model studies, showing that early re-epithelialization and faster wound closure result from accelerated collagen accumulation induced by decreasing of MMP2 and MMP9 expression (Li et al. 2017). Furthermore, MMP downregulation is a relevant explicative mechanism involved in the efficacy of the Food and Drug Administration – approved MMP inhibitor currently used to treat periodontal disease (Golub et al. 1998; Ashley 1999; Preshaw et al. 2004; Boelen et al. 2019). Such reports demonstrate clearly that the downregulation of some MMPs could favour the re-epithelialization while limiting tissue infiltration by the inflammatory cells (Li et al. 2017, p.). Note that other cellular types such as mesenchymal and cancer cells can secret MMPs to promote tissue infiltration and invasion (Fields 2019). Otherwise, in addition to an ECM degradation, MMPs act through other mechanisms in several processes involved in the tissue repair, including inflammation, re-epithelialization, angiogenesis and remodelling of new ECM (Xue et al. 2006). In vivo studies should bring more information regarding the effect of BW. Nevertheless, the overall responses of TGFβ, IL6, TNFα, MMP, at the protein level, and of ABCA1, ABCG1 and IDOL, at the mRNA level, prompt us to hypothesize a putative modulation of the transcriptional activity of the liver X receptors (Fowler et al. 2003; Chang et al. 2008; Ouedraogo et al. 2017) and a possible interference with the management of the oxidative stress.

Our data points out that the thermal spring water from La Bourboule could improve wound healing by controlling keratinocyte physiology (graphical abstract) although an in vitro model does not fully replicate all the complex interactions functioning in vivo. One of the mechanisms of BW could be the modulation of various inflammation regulators at the mRNA and/or protein levels. The effect of BW, as shown in this study, begins within the 24h of action, meaning potential positive outcomes at the early steps of wound healing, when inflammation should be controlled but not completely blocked to allow both immune activation and controlled re-epithelialization. It may be relevant to explore in vivo long term effect of BW in further studies. Besides, more experiments are required to identify any biological active compound within BW and all its putative effects in healthy or disease conditions.

Funding: “This study was supported by grants from CNRS, INSERM, Université Clermont Auvergne, Auvergne-Rhône-Alpes Region, La Régie Municipale des Grands Thermes, European Regional Development Fund and Greentech SA (Saint-Beauzire, France)”.

Competing Interests: “E. FILAIRE was employed by GREENTECH and J-Y. BERTHON is founder of GREENTECH SA. This does not alter authors’ adherence to all Exposure and Health policies on sharing data and materials. The other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Author Contributions: Zangbéwendé Guy Ouédraogo, Jean-Yves Berthon, Anne Fogli, Vincent Sapin, Silvère Baron and Jean-Marc A. Lobaccaro contributed to the study conception and design. Material preparation and data collection were performed by Zangbéwendé Guy Ouédraogo, Anne Fogli, Allan Fouache, Amalia Trousson, Silvère Baron and Jean-Marc A. Lobaccaro. All authors contributed to data analysis. The project was administrated and supervised by Silvère Baron and Jean-Marc A. Lobaccaro. The first draft of the manuscript was written by Zangbéwendé Guy Ouédraogo, Anne Fogli, Allan Fouache and Jean-Marc A. Lobaccaro and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability Statement: All data are available in our paper and in the supplementary files. Additional information could be provided by the corresponding authors on reasonable request.

Acknowledgments: We are grateful to Dr. Simon BROAD (Centre for Stem Cells & Regenerative Medicine, King's College London, UK), Pr. Andrei TCHIRKOV, Dr. Aurelie VEGA, Ms. Sandra CARLET-DOLLET (CHU Clermont-Ferrand) for the material supply. Our thanks go also to Gaston OUEDRAOGO for proofreading in English. ZGO was an associate researcher funded by Auvergne-Rhône-Alpes Region and European Regional Development Fund.

Ethic Statements: The investigations were performed in compliance with national and local legislations. The collection and the analyses were conducted with approval of the managers of the springs of La Bourboule. The reported data did not involve either clinical investigation in human or animal experimentation.

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No competing interests reported.

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Modulation of wound healing regulators by thermal spring water from La Bourboule

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Cell culture

2.2. Thermal spring water analysis and preparation

2.3. Cell counting assay

2.4. In vitro wound healing assay

2.5. RNA purification and real-time PCR

2.6. Cytokine and metallopeptidase Luminex assay

2.7. Statistics

3. Results

3.1. Physical and chemical characteristics of the Thermal spring water from La Bourboule

3.2. Effect of the thermal spring water of La Bourboule on HaCaT cell growth

3.3. Thermal spring water of La Bourboule upregulates the TGFβ pathway and downregulates the MMP release

4. Discussion

5. Conclusions

Statements and Declarations

References

Additional Declarations

Supplementary Files

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