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International Journal of Hyperthermia
ISSN: 0265-6736 (Print) 1464-5157 (Online) Journal homepage: http://www.tandfonline.com/loi/ihyt20
Innate/inflammatory bioregulation and clinical
effectiveness of whole-body hyperthermia
(balneotherapy) in elderly patients with
osteoarthritis
Isabel G álvez, Silvia Torres-Piles & Eduardo Ortega
To cite this article: Isabel G álvez, Silvia Torres-Piles & Eduardo Ortega (2018): Innate/
inflammatory bioregulation and clinical effectiveness of whole-body hyperthermia
(balneotherapy) in elderly patients with osteoarthritis, International Journal of Hyperthermia, DOI:
10.1080/02656736.2018.1502896
To link to this article: https://doi.org/10.1080/02656736.2018.1502896
© 2018 The Author(s). Published with
license by Taylor & Francis Group, LLC.
Published online: 08 Oct 2018.
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Innate/inflammatory bioregulation and clinical effectiveness of whole-body
hyperthermia (balneotherapy) in elderly patients with osteoarthritis
Isabel G /C19alveza, Silvia Torres-Pilesband Eduardo Ortegaa
aResearch Group in Immunophysiology, Faculty of Sciences, Department of Physiology, University of Extremadura, Badajoz, Spain;bResearch
Group in Immunophysiology, Faculty of Medicine, Department of Medical-Surgical Therapy, University of Extremadura, Badajoz, Spain
ABSTRACT
Objectives: Balneotherapy with mud application (mud therapy) is a common hydrothermal interven-
tion for the treatment and rehabilitation of elderly patients with osteoarthritis, leading to anti-inflam-matory effects. The main purpose of this investigation was to study a role for regulatory T cells in
these anti-inflammatory effects. The second objective was to assess whether the neutrophil-mediated
innate response is affected by these anti-inflammatory effects.
Methods: Thirty-six elderly patients with knee osteoarthritis underwent a 10-day cycle of balneother-
apy at a spa center. They received daily sessions of whole-body mud therapy at 40 –42
/C14C, using min-
eral-medicinal water and mud. IL-8 and TGF- bserum concentrations, percentage of circulating CD4ț
CD25țFOXP3țand CD8țCD28–regulatory T cells, and neutrophil phagocytic capacity were eval-
uated at baseline and at the end of the intervention. Clinical assessments included knee flexion andextension angle, pain, stiffness, physical function and health-related quality of life.
Results: All clinical outcomes significantly improved. Circulating concentrations of IL-8 and TGF- b
decreased, which correlated with decreased pain and improved knee flexion, respectively. Percentage
of CD4țregulatory T cells decreased, whereas CD8țregulatory T cells increased. Neutrophil functional
capacity increased.Conclusions: Balneotherapy with mud application was effective in the management of osteoarthritis
symptoms. The anti-inflammatory effect mediated by cytokines contributed to the improvement in
pain and joint function; and changes in the circulating percentage of regulatory T cells seem to be
involved in the anti-inflammatory effects. Improvement in neutrophil function after mud therapy
reflects an optimal bioregulatory effect on the inflammatory and innate responses.ARTICLE HISTORY
Received 11 May 2018
Revised 21 June 2018
Accepted 15 July 2018
KEYWORDS
Knee osteoarthritis; mudtherapy; granulocytes;cytokines; regulatory T cells;inflammation
Introduction
Osteoarthritis (OA) is the most common arthritic disease and
a leading cause of pain, disability and impaired quality of life
worldwide, especially among the elderly. It affects synovial
joints (mainly knees and hips), in which cartilage destruction,
subchondral bone remodeling, osteophyte formation and
synovial inflammation is found, leading to joint stiffness,
swelling, pain and loss of mobility [ 1,2]. Although the multi-
factorial pathophysiological mechanisms of OA still remain
largely unknown, it is known that various inflammatory and
immune processes are strongly implicated in the pathogen-
esis, progression and burden of OA [ 3]. In addition to local
inflammatory events occurring within joint tissues such as
the release of inflammatory mediators by cartilage, bone and
synovium [ 4,5], low-grade systemic inflammation also plays a
pivotal role in this condition. The presence of low-grade sys-
temic inflammation could lead to the initiation and aggrava-
tion of OA, whereas locally produced inflammatory
mediators, such as cytokines, might be reflected in peripheral
blood and contribute to perpetuate this systemicinflammatory status [ 6–8]. Patients with OA also present
changes in peripheral blood T cell composition [ 9].
Furthermore, they have an immune-neuroendocrine dysregu-lation affecting the negative feedback between the inflam-matory and stress responses along with the decreased
functional capacity of phagocytes, thus reflecting an altered
bioregulation of the innate/inflammatory responses andsuppression of the immune system ’s defenses against
pathogens [ 8].
Traditional pharmacologic therapies for OA focus on treat-
ing inflammation (e.g., NSAIDs, corticosteroids) but theymerely palliate the symptoms rather than modifying thecourse of the disease. Besides, pharmacologic and surgicaltreatments are associated with problems including invasive-ness, the high cost of the procedures and, frequently,adverse events [ 10]. Therefore, there is a need for developing
conservative therapies for OA focusing on evidence-basedmechanisms of effectiveness. Balneotherapy is the set of
methods and practices which, based on scientific evidence,
use medically and legally recognized mineral-medicinal
CONTACT Eduardo Ortega orincon@unex.es Research Group in Immunophysiology, Faculty of Sciences, Department of Physiology, University of
Extremadura, Avda. Elvas s/n, Badajoz 06071, Spain
/C2232018 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0/ ), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.INTERNATIONAL JOURNAL OF HYPERTHERMIA
https://doi.org/10.1080/02656736.2018.1502896

waters, muds and natural gases from natural springs for
therapeutic purposes inside the facilities of thermal spa cen-ters. Particularly, muds are maturated muddy suspensions
composed of a complex mixture of fine-grained materials of
geologic origin, mineral water and common organic com-
pounds from the biological metabolic activity. Thus, mud
therapy is a balneological intervention that consists of theexternal application of mud for therapeutic purposes [ 11].
Temperature has a central role in the effects of these thera-
pies. Mineral-medicinal water and mud are generally appliedhot as they are excellent vehicles for the transference of
heat (being able to hold heat and release it slowly) so these
treatments can be considered thermotherapeutic interven-tions [ 12]. The peculiarity of balneotherapy is that its benefi-
cial effects on the organism are brought about not only by
the physical properties of mineral-medicinal water and mud
but also by their chemical and biological composition
[11,13]. Balneotherapy, without or with mud application, is a
common non-pharmacological approach for OA carried out
by physiotherapists and physicians (specialists in Physical
Medicine and Rehabilitation, and in Medical Hydrology). Inthis context, balneotherapy could not only reduce pain and
stiffness, and improve joint mobility and quality of life, but
also effectively prevent or delay the progression of OA [ 14].
Several studies have reported the beneficial effects of
balneotherapy [ 15–18], and particularly mud therapy, on
function, perceived pain, analgesic drug consumption, and
quality of life in patients with OA, suggesting that they are
effective and safe alternatives in the clinical management ofthis pathology [ 14,19,20]. Moreover, this strategy is also cost-
effective, making it a relevant part of the public health sys-
tems of many countries within and outside Europe [ 21,22].
However, despite the evidence of clinical and symptomatic
benefits of these therapies, their role in modern medicine is
still under discussion [ 16], mainly because the biological and
physiological mechanisms underlying these benefits have
not yet been completely elucidated. It is then necessary toachieve a deeper understanding of these biological mecha-
nisms of effectiveness, so balneotherapy can be practiced by
health professionals based on scientific evidence that sup-ports its use in patients with OA. In this context, our group
has recently found systemic cytokine-mediated anti-inflam-
matory effects together with a better immune-neuroendo-
crine regulation after mud therapy in patients with OA,
which constitutes a mechanism of effectiveness responsiblefor the amelioration of the symptoms in this pathology [ 14].
However, to the best of our knowledge, nothing is known
about the effects of mud therapy on the cellular immuneresponse; particularly those involved in the innate/inflamma-
tory response.
Thus, our main objective was to evaluate the participation
of regulatory T (Treg) cells in the anti-inflammatory effects of
mud therapy in elderly OA patients. In contrast, and taking
into account our previous observations of a reduced neutro-
phil-mediated innate immune response in patients with OA
[8], the second objective was to assess whether the innate
immune cell function is affected by the anti-inflammatory
effects of this intervention. In addition, we examined thecorrelation between concentrations of systemic inflammatory
cytokines and clinical outcomes of the OA patients after theintervention.
Methods
This was a prospective, controlled study conducted at thehealthcare and spa center ‘El Raposo ’(Puebla de Sancho
P/C19erez, Badajoz, Spain), declared a Public Utility in 1926. We
evaluated the effects of a cycle of mud therapy, using mudnaturally found at the mineral-medicinal spring, in a group
of patients with OA participating in the Social Thermalism
Programme organized by the Elderly and Social ServicesInstitute of Spain ’s Ministry of Health, Social Policy,
and Equality.
The volunteers assessed for eligibility in the study were
68 patients with primary knee OA. After being informed ofthe investigation, all volunteers from this homogenous groupthat complied with the inclusion and exclusion criteria wereaccepted. The inclusion criteria were to be /C2160 years old
and to be diagnosed with primary knee OA by a rheumatolo-
gist following American College of Rheumatology (ACR) crite-ria [ 23]. The exclusion criteria were having any infection,
neoplastic illness, or cardiopulmonary, vascular, inflammatory,immune or other musculoskeletal condition, having had a
total or partial knee replacement, having received any phys-
ical therapy in the previous six months, having consumedNSAIDs in the previous three days, having received intra-articular injections of corticosteroids or hyaluronic acid in theprevious six months, or having received oral or local cortico-
steroid or anticytokine therapy that could influence inflam-
matory and immune parameters. Forty-two patients satisfiedthe eligibility criteria and were included in the study. Sixpatients were lost to follow-up, leaving 36 patients from 62to 80 years in age (mean age ± SEM 70.71 ± 1.05 years) forevaluation ( Figure 1 ). All patients signed written informed
consent prior to inclusion in the study. Each participant was
Figure 1. Flowchart of patients through the study.2 I. G/C19ALVEZ ET AL.

identified with an alphanumeric code in order to preserve
their anonymity.
Table 1 shows baseline and posttreatment anthropometric
measurements along with demographic data. These parame-
ters and other clinical variables were evaluated along with
blood sampling between 8 am and 9 am. Baseline evaluation
and sampling were performed one day after arrival at the
spa center, before initiating the first session of mud therapy.
Posttreatment sampling was carried out a day after the last
session of mud therapy, thus avoiding the evaluation of the
effect of an acute intervention.
The study was approved by the Ethical Committee of the
University of Extremadura, Spain, in accordance with the
guidelines of the European Community Council Directives
and the Helsinki Declaration.
Intervention
The spring water of the spa center ‘El Raposo ’emerges at
16.5/C14C, forming a natural stream where the mud is obtained.
Then, the mud is placed in a maturation tank along with the
mineral-medicinal water and left to mature until it reaches
optimal biological, chemical and thermophysical properties
for the application.
The water of ‘El Raposo ’spa contains bicarbonate
(396.5 mg/L) and calcium (130.2 mg/L) as predominant ions.The mineral-medicinal water fraction represents /C2440% of the
mud. Mud ’s solid content ( /C2460%) consists of a mixture of
silt, clay and sand. It is basically composed of phyllosilicates
(smectite and illite), quartz and calcite. The major chemical
elements of the mud are SiO
2, CaO, Al 2O3and Fe 2O3[24,25].
The predominant microalgae species present in the mud was
identified morphologically as Monoraphidium pusillum
(Spanish Bank of Algae).
Patients received a daily session of mud therapy for 10
consecutive days, according to the therapeutic procedure
implemented by their healthcare professionals. The interven-
tion was always conducted in the morning by the samephysiotherapists. The sessions consisted of the whole-body
application of mud at 40 –42
/C14C by brush, followed by a dry-
ing period of 45 –60 minutes in a solarium. Subsequently,
patients received a mud bath (consisting of mineral-medi-
cinal water and mud) at 38 –40/C14C for 15 minutes. Finally,
mud remnants were removed with a thermal water jet
(38 –40/C14C) for 2 minutes. No adverse events were reported.Anthropometric and clinical data
Body weight, body mass index (BMI), waist-hip ratio (WHR)
and knee flexion and extension angle were measured usingstandardized methods.
Perceived pain intensity was assessed using a visual ana-
logue scale (VAS) [ 26]. Besides, Western Ontario and
McMaster Universities Arthritis Index (WOMAC) (5-point Likertformat) were used to evaluate pain, stiffness, and physicalfunction associated with OA [ 27]. The EuroQol-5D (EQ-5D)
questionnaire was used to measure the generic health-related quality of life [ 28].
Whole blood and serum isolation
In a fasted state, peripheral blood samples were drawn by an
antecubital fossa vein sterile puncture and collected inserum-separating and heparinized tubes. Blood samples forserum isolation were maintained for 15 –20 minutes at room
temperature after extraction to allow clotting. Then, theywere centrifuged at 700 gfor 10 min, and the serum samples
were aliquoted and stored at –80
/C14C until assay.
Determination of systemic concentration of IL-8 and
TGF-b
Serum inflammatory cytokine concentrations (IL-8 and TGF- b)
were measured with the Bio-PlexVR200 system and LuminexVR
xMAP technology (Bio-Rad Laboratories, Inc., Hercules, CA)using a high sensitivity kit (Bio-Techne, R&D Systems, Inc.,Minneapolis, MN). To avoid inter-assay variations, all sampleswere analyzed with the same kit on the same day.
Regulatory T cell phenotyping
Blood samples were fixed and permeabilized using the InsideStain Kit (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany),
following manufacturer instructions. Then, CD3-FITC, CD4-APC
Vio770, CD25-APC, CD28-PE Vio770, CD8-PerCP and FOXP3-PEantibodies (Miltenyi Biotec GmbH) were added to 100 lLo f
permeabilized samples in the corresponding fluorescenceorder in several tubes. After 1 hour of incubation at room tem-perature, 1 mL of diluting buffer from intra staining kit wasadded and samples were spun down. Pellet was resuspendedin 500 lL of PBS and samples were analyzed using a
MACSQuant
VRAnalyzer 10 (Miltenyi Biotec GmbH) flow cytom-
eter. Twenty thousand events were acquired per sample.
Study of the neutrophils ’phagocytic capacity
The neutrophils ’phagocytic capacity against opsonized bac-
teria ( Staphylococcus epidermidis ) was evaluated by flow
cytometry of heparinized whole blood. This quantitativetechnique allows the evaluation of the percentage of active‘phagocytic neutrophils ’and the number of bacteria ingested
per cell by measuring the mean fluorescence intensity (MFI)of active phagocytic cells, which reflects the phagocyticactivity of neutrophils [ 8].Table 1. Anthropometric and demographic data.
Pre-treatment ( n¼36) Post-treatment ( n¼36)
Sex (women/men) 23/13 –
Age (years) 70.71 (±1.05) –
Weight (kg) 75.22 (±2.25) 75.75 (±2.23)BMI (kg/m2) 28.15 (±0.89) 28.35 (±0.88)
WHR 0.90 (±0.01) 0.90 (±0.01)
Employment status
Blue collar n¼12 (33.33%) –
White collar n¼5 (13.88%) –
Unemployed n¼19 (52.77%) –
Data are expressed as mean (± SEM)
BMI: body mass index; WHR: waist-hip ratio.INTERNATIONAL JOURNAL OF HYPERTHERMIA 3

In brief, bacteria were stained with fluorescein isothio-
cyanate (FITC) (1 lg/ml) and opsonized with human serum.
Aliquots of 200 ll of blood from each donor were incubated
(for 1 h at 37/C14C in the dark with shaking) with 50 ll of the
opsonized bacteria, 10 lg/ml of Hoechst 33342, 1 lg/ml of
7-actinomycin-D (7AAD), 250 ll of PBS and 2% of fetal bovine
serum (FBS). The controls consisted of 100 ll of blood com-
bined with 10 lg/ml of Hoechst 33342, 1 lg/ml of 7AAD, 400 ll
of PBS and 2% FBS. Blood samples were then analyzed with aflow cytometer (MACSQuant
VRVYB; Miltenyi Biotec GmbH).
Statistical analysis
The values are expressed as mean ± standard error of themean (SEM). The normal distribution of the variables waschecked using the Kolmogorov –Smirnov normality test, fol-
lowed by Student ’st-test for paired samples and Pearson
correlation coefficient for correlation analysis. The signifi-cance level was set at p<.05. Calculations were performed
using the IBM
VRSPSSVRStatistics version 22 software package
(IBM Corp., Armonk, NY).
Results
Clinical outcomes: function, pain, stiffness and
quality of life
After the cycle of mud therapy, knee flexion and extension
angle significantly increased; perceived pain (VAS) and OA-related pain, stiffness and physical function (WOMAC index)
significantly improved; and health-related quality of life (EQ-
5D) significantly improved in the OA patients (questionnaireresponse rate 93%). The detailed results and statistical signifi-
cance are presented in Table 2 .
Systemic inflammatory cytokine concentrations:
correlation with clinical outcomes
Circulating concentrations of IL-8 and TGF- bwere markedly
decreased after the intervention (IL-8, p<.001; TGF- b,
p<.01) ( Table 2 ). Using these values, an inflammatory index
was calculated, indicating the pro-inflammatory cytokine IL-8
to the anti-inflammatory cytokine TGF- bratio. This inflamma-
tory index decreased after the intervention in all groups,thus showing a greater decrease in IL-8 levels than in those
of TGF- b(Table 2 ).
In posttreatment state, a significant positive correlation
(p<.01) between IL-8 serum concentration and perceived
pain, as well as a significant negative correlation ( p<.01)
between TGF- band knee flexion angle were found
(Figure 2 ).
CD41CD251FOXP31and CD81CD28–regulatory
T cells phenotypic analysis
Flow cytometer phenotypic analysis revealed that the per-
centage of CD4țCD25țFOXP3țTreg cells was significantly
reduced after mud therapy ( p<.01) ( Figure 3(A) ), whereas
CD8țCD28–Treg cell post-intervention percentage notably
increased ( p<.001) ( Figure 3(B) ).
Neutrophil phagocytic capacity
Figure 4 shows the results relating to the neutrophil func-
tional activity. Both the percentage of ‘phagocytic neu-
trophils ’(%) ( Figure 4(A) ,p<.01) and the phagocytic activity
(MFI) ( Figure 4(B), p<.001) of circulating neutrophils against
opsonized bacteria significantly increased after mud therapyin the patients with OA.
Discussion
Balneotherapy with mud application is a well-tolerated, cost-effective, thermal treatment frequently used for rheumaticdiseases, especially knee OA, reinforcing the suitability of thistherapy in health systems [ 21]. The clinical effectiveness of
this therapy in OA has been largely demonstrated by numer-ous investigations [ 29,30], including studies from our group
in the same spa center as the present investigation [ 14,19].
Results presented here corroborated the clinical benefits of
this therapy in OA patients, as manifested in the improve-
ment in knee flexion and extension angle, perceived pain,stiffness, functional capacity and health-related quality of lifeafter the cycle of mud therapy, thus confirming that it iseffective in the management of OA. However, the biologicalmechanisms of action still remain unknown. Recently, a num-ber of studies have focused on assessing the influence ofmud therapy on different biomarkers [ 14,30–32], includingTable 2. Clinical measurements and serum concentration of inflammatory cytokines IL-8 and TGF- bin
patients with OA before (pretreatment) and after (posttreatment) mud therapy intervention.
Pretreatment Posttreatment
Knee flexion angle (/C14) 104.22 (±2.40) 115.38 (±2.71) /C3/C3/C3
Knee extension angle (/C14) 171.72 (±1.74) 176.11 (±1.27) /C3/C3
VAS 5.93 (±0.39) 3.48 (±0.58)/C3/C3/C3
WOMAC 39.08 (±2.63) 25.51 (±2.81)/C3/C3/C3
EQ-5D 7.64 (±0.26) 6.58 (±0.29) /C3/C3/C3
IL-8 (pg/mL) 409.37 (±81.44) 120.66 (±18.05) /C3/C3/C3
TGF-b(pg/mL) 93 746.9 (±10 835.9) 78 288.9 (±8036.67)/C3/C3
Inflammatory index (IL-8/TGF- b)(/C210-3) 4.3 (±0.6) 2.1 (±0.3)/C3/C3/C3
Data are expressed as mean (±SEM)
VAS: visual analog scale; WOMAC: Western Ontario and McMaster Universities Arthritis Index; EQ-5D:
EuroQol-5D.
/C3/C3p<.01
/C3/C3/C3p<.001 with respect to basal values.4 I. G/C19ALVEZ ET AL.

novel biomarkers such as microRNA [ 33], due to the import-
ance of ascertaining the mechanisms of effectiveness of thistherapy so it can be prescribed, carried out, funded andregulated according to scientific evidence. Particularly, our
group has shown that an immune-neuroendocrine regulation
together with a systemic cytokine-mediated anti-inflamma-
tory effect constitutes an immunophysiological mechanism
of action that underlies the clinical benefits [ 14]. In the pre-
sent work, we have assessed the circulating concentrationsof two inflammatory cytokines: the pro-inflammatory IL-8
and the anti-inflammatory TGF- b. Both have been reported
to be elevated systemically in OA patients [ 8,34], contribu-
ting to the chronic low-grade inflammatory status present in
this pathology [ 8]. This is particularly notable for IL-8 sys-
temic concentrations, as they have been found to be dramat-
ically elevated by 30-fold in OA patients compared withhealthy individuals [ 8]. Results clearly showed that mud ther-
apy induced a decline in the high systemic levels of both
cytokines in OA patients, thus confirming a global anti-
inflammatory effect of the intervention [ 14]. Furthermore, as
the inflammatory response must be considered as a whole,in the context of a global anti-inflammatory response, a
good balance between systemic pro- and anti-inflammatory
cytokines is also important [ 35]. Thus, the inflammatory index
(IL-8/TGF- bratio) decreased, revealing a greater decline in
pro-inflammatory IL-8 levels than in those of TGF- b. It is well
known that IL-8 contributes to joint pain and hyperalgesia
due to its pro-inflammatory and proalgesic effects [ 36]; and
TGF-b, despite being classically considered an anti-inflamma-
tory cytokine, has a dual role in OA as a result of its anti-
inflammatory/anabolic and pro-inflammatory/catabolic effects
in the joint, the latter characterized by osteophyte formation
Figure 2. Correlation between serum inflammatory cytokine concentrations and clinical outcomes in patients with OA after the cycle of mud therapy: IL-8 and
perceived pain (A) and TGF- band knee flexion angle (/C14) (B). VAS: visual analog scale.
Figure 3. Percentage of CD4țCD25țFOXP3țTreg cells (A) and CD8țCD28–Treg cells (B) in patients with OA before (pre-treatment) and after (post-treatment)
the cycle of mud therapy. Columns represent the mean ± SEM of independent assays performed in duplicate for each participant. /C3/C3p<.01,/C3/C3/C3p<.001 with
respect to basal values.
Figure 4. Functional capacity of circulating neutrophils in patients with OA
before (pretreatment) and after (posttreatment) the cycle of mud therapy: per-
centage of ‘phagocytic neutrophils ’(A) and phagocytic activity of circulating
neutrophils (B). Columns represent the mean ± SEM of independent assays per-formed in duplicate for each participant. /C3/C3p<.01;/C3/C3/C3p<.001 with respect to
basal values.INTERNATIONAL JOURNAL OF HYPERTHERMIA 5

and cartilage damage [ 4,37]. Indeed, IL-8 levels were posi-
tively correlated with VAS pain score, and TGF- blevels were
negatively correlated with knee flexion angle after the inter-
vention, showing that those patients with lower IL-8 and
TGF-blevels presented lower pain scores and greater knee
flexion angles, respectively. Therefore, these results
altogether confirmed that the reduction of systemic inflam-
mation in OA after mud therapy constitutes a mechanism ofeffectiveness that contributes to the amelioration of painand impairment.
To the best of our knowledge, the present work is the
first to assess Treg-mediated inflammatory response inbalneotherapy in general and, particularly, in mud therapy.
Although their frequency and function in OA has not yet
been clearly elucidated, the importance of Treg cells asimmunoregulators in inflammatory pathologies has been
highlighted in recent studies, suggesting the interest of
developing new therapeutic approaches based on themanipulation of Treg cell responses [ 9,38]. Both CD4
țCD25ț
FOXP3țand CD8țCD28–Treg cells are essential in regulat-
ing immune reactions, preventing autoimmunity, moderatinginflammation and maintaining immune homeostasis. They do
so through several complex mechanisms, including suppres-
sion of aberrant or excessive immune responses by alteringthe functional status of immune cells [ 38–40]. Our results
indicate that the increase in CD8
țTreg cells in OA patients
after mud therapy seems to be a mechanism contributing toreduce the inflammatory status. In fact, patients with differ-ent immune and inflammatory diseases have lower numbers
of or less functional CD8
țTreg cells [ 38]. In this context,
induction of CD8țTreg cells has shown to have beneficial
anti-inflammatory effects, effectively downregulating exces-
sive inflammatory activity [ 38,41]. Surprisingly, CD4țTreg
cells percentage decreased after the cycle of mud therapy inOA patients. There have been previous observations of raised
levels of circulating CD4
țTreg cells in OA [ 42–44], but para-
doxically, despite elevated numbers of these cells, inflamma-tion is still present in this pathology. This can be understood
as an attempt of the immune system to control the inflam-
matory responses, but the persistence of inflammation couldindicate that these cells present functional impairment that
limits their capacity to suppress ongoing disease, perhaps
due to an inhibition of their suppressive functions by pro-inflammatory cytokines or because of an increased number
of activated effector T cells [ 44,45]. In this way, the decline in
CD4țTreg cell numbers after mud therapy could be a
reflection of the anti-inflammatory effects of this therapy, sothe reduction in the inflammatory status could explain a
lesser need of CD4 țTreg cells to counterbalance the exces-
sive inflammatory responses in OA. As far as we know, thisreport is the first in evaluating Treg cells in the context of
the anti-inflammatory effects of balneotherapy, hence, unfor-
tunately, we cannot discuss the results in relation to anyother similar investigations.
Patients with OA present a reduced phagocytic activity of
circulating neutrophils, and thus could be more susceptibleto infection than healthy individuals [ 8]. In this context, the
question arising now was: could the anti-inflammatoryeffects of mud therapy induce further suppression of the
immune system ’s defenses against pathogens? Results clearly
showed that not only did the functional capacity of neutro-
phils not decrease any further, but instead it increased sig-
nificantly, in accordance with previous results in micemacrophages after hydrotherapy [ 46]. This improved func-
tional activity of neutrophils after the intervention reflects a
greater defense capacity against pathogen challenge andthus a potential lower susceptibility to infections. Bearing inmind that stress-induced physiological concentrations of glu-
cocorticoids mediate the stimulation of some of the innate
immune system ’s functions, including phagocytosis [ 47,48],
and that previous investigations from our research group
found an increase in the systemic concentrations of cortisol
after the same balneological intervention evaluated in thepresent study [ 14], it is plausible to hypothesize that this
stress hormone could be mediating the stimulation of the
phagocytic activity of neutrophils in OA patients afterbalneotherapy [ 12]. Further in vitro studies will be needed to
confirm this hypothesis. This improvement in the neutrophil-
mediated innate immune response together with the prom-inent anti-inflammatory effects observed in the present study
and in our previous one [ 8] are suggestive of a ‘bioregulatory
effect of balneotherapy ’, a term introduced in other strat-
egies, such as exercise, for treating low-grade inflammatory
pathologies; consisting in a reduction in unhealthy inflamma-
tory biomarkers together with optimal innate response suchas phagocytic activity [ 49]. We believe this constitutes an
interesting novel clinical aspect of the therapeutic effects of
mud therapy.
It is noteworthy that although it has been reported that
the main therapeutic effects are due to the elevated tem-
perature of application, the chemical and biological compo-
nents of mineral-medicinal water and mud could cross theskin barrier and play a part in these effects as well [ 12,13], so
it is plausible to think that the mechanism of action probably
results from a complex synergistic combination of both fac-tors [ 12,30]. In this regard, further studies seem to be neces-
sary to ascertain the ideal temperature to elicit optimal
therapeutic responses without causing damaging effects. Inaddition, future studies looking deeper into the cellular and
molecular mechanisms of action that mediate the role of IL-8
could also be relevant. In this context, results of the presentinvestigation are in agreement with the increase in systemic
concentrations of cortisol previously reported in OA patients
undergoing balneotherapy [ 14], as increased physiological
concentrations of cortisol induce a decrease in the release ofinflammatory cytokines from immune cells [ 50
] and thus may
contribute to decrease the magnitude of the inflammatory
response after balneotherapy interventions [ 12]. Thus, further
in vitro studies evaluating the effect of cortisol on inflamma-
tory cells from OA patients could clarify this interaction.
Furthermore, studies focusing on the role of prostaglandinsin mediating the hyperalgesic effects of IL-8 in OA patients
(and its reduction after balneotherapy) may be, in our opin-
ion, especially relevant.
In conclusion, results suggest that balneotherapy, particu-
larly mud therapy, is an effective complementary approach6 I. G/C19ALVEZ ET AL.

in the management of OA symptoms. Its anti-inflammatory
effect, which is mediated by systemic inflammatory cyto-
kines, contributed to the clinical improvement in pain and
joint function; and changes in the percentage of circulating
regulatory T cells seem to be also clearly involved in this
anti-inflammatory effect. Moreover, neutrophil functional
capacity increased after mud therapy, thus reflecting an opti-
mal bioregulatory effect on the inflammatory and
innate responses.
Acknowledgments
We are grateful to the Facility of Bioscience Applied Techniques (STAB,
University of Extremadura, Spain) and the spa center ‘El Raposo ’for
technical and human support.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was partially supported by the Gobierno de Extremadura-
FEDER under grant GR 15041, EE-14-0082-4. IG is recipient of a
‘Formaci /C19on del Profesorado Universitario (FPU) ’pre-doctoral contract
under grant FPU15/02395 from the Ministerio de Educaci /C19on, Cultura y
Deporte, Spain.
ORCID
Isabel G /C19alvez http:orcid.org/0000-0002-4294-4507
Eduardo Ortega http:orcid.org/0000-0002-7007-7615
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