nutrients

Article
Dietary Intake of n-3 PUFA-Enriched Hen Eggs Changes
Inflammatory Markers’ Concentration and Treg/Th17 Cells
Distribution in Blood of Young Healthy Adults—A
Randomised Study
Nikolina Kolobarić 1,2 , Ines Drenjančević 1,2,† , Anita Matić 1,2 , Petar Šušnjara 1,2 , Zrinka Mihaljević 1,2
and Martina Mihalj 1,2,3, *

1 Department of Physiology and Immunology, Faculty of Medicine Osijek, Josip Juraj Strossmayer University
of Osijek, J. Huttlera 4, 31000 Osijek, Croatia; nbdujmusic@mefos.hr (N.K.); ines.drenjancevic@mefos.hr (I.D.);
amatic@mefos.hr (A.M.); psusnjara@mefos.hr (P.Š.); zmihaljevic@mefos.hr (Z.M.)
2 Scientific Center of Excellence for Personalized Health Care, Josip Juraj Strossmayer University of Osijek,
Trg Svetog Trojstva 3, 31000 Osijek, Croatia
3 Department of Dermatology and Venereology, Osijek University Hospital, J. Huttlera 4, 31000 Osijek, Croatia
* Correspondence: mmihalj@mefos.hr; Tel.: +385-3151-2800
† Principal investigator.

Abstract: In the present study, we aimed to determine the effects of n-3 polyunsaturated acid (PUFA)



supplementation (~1053 mg/per day), i.e., α-linolenic (~230 mg), eicosapentaenoic (~15 mg), and


docosahexaenoic acid (~105 mg), through hen eggs, on pro- and anti-inflammatory parameters in
Citation: Kolobarić, N.; healthy individuals (23.8 ± 2.57 years old). Here, we demonstrate differential effects of regular hen
Drenjančević, I.; Matić, A.; Šušnjara, eggs (N = 21; W/M = 10/11) and n-3 PUFA-enriched hen eggs (N = 19; W/M = 10/9) consumption
P.; Mihaljević, Z.; Mihalj, M. Dietary on the serum levels of lipid mediators, representation of peripheral T helper cell subsets (recently
Intake of n-3 PUFA-Enriched Hen
activated T-helper cells, nTreg, Th17 and non-Th17-IL-17A secreting T-helper lymphocytes) and
Eggs Changes Inflammatory Markers’
their functional capacity for cytokine secretion. Both diets significantly altered systemic levels
Concentration and Treg/Th17 Cells
of pro-inflammatory and inflammation resolving lipid mediators; however, only the n-3 PUFAs
Distribution in Blood of Young
Healthy Adults—A Randomised
group showed a significant shift towards anti-inflammatory prostanoids and increased levels of pro-
Study. Nutrients 2021, 13, 1851. resolving oxylipins. Both study groups showed reduced frequencies of peripheral nTreg lymphocytes
https://doi.org/10.3390/nu13061851 and decreased rates of peripheral Th17 cells. Their functional capacity for cytokine secretion was
significantly altered only in the n-3 PUFAs group in terms of increased transforming growth factor
Academic Editor: A. Catharine Ross β-1 and reduced interleukin 6 secretion. Diet supplemented with n-3 PUFAs alters immune response
towards inflammation resolving conditions through effects on lipid mediators and cytokine secretion
Received: 24 April 2021 by T lymphocytes in human model without underlying comorbidities.
Accepted: 26 May 2021
Published: 28 May 2021 Keywords: fatty acids; dietary supplements; eicosanoids; inflammation; T lymphocytes

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1. Introduction
iations.
The finely tuned process of inflammation consists of several steps, including im-
mune cell activation, recruitment of the cells to the site of inflammation, the release of
inflammatory mediators, and increase in vascular permeability leading to resolution of
the inflammatory process and healing [1]. Failure of the immune system to mitigate and
Copyright: © 2021 by the authors.
efficiently resolve inflammation leads to the chronic condition of low-grade inflammation,
Licensee MDPI, Basel, Switzerland.
which underlies the development of chronic diseases such as cardiovascular (CVD) and
This article is an open access article
metabolic diseases [2]. Considering that, there is a certain responsibility for finding con-
distributed under the terms and
venient tools that could improve the rate of efficient resolution in the context of reduced
conditions of the Creative Commons
Attribution (CC BY) license (https://
inflammation conditions.
creativecommons.org/licenses/by/
In adaptive immune response to noxis (infection; mechanical, thermal, or chemical
4.0/). stimuli), several classes of CD4+ effector T lymphocytes are differentiated from naïve

Nutrients 2021, 13, 1851. https://doi.org/10.3390/nu13061851 https://www.mdpi.com/journal/nutrients

Nutrients 2021, 13, 1851 2 of 20

T cells, including T helper 17 lymphocytes (Th17) and the T regulatory lymphocytes

(Tregs) [3–5]. The interplay between pro-inflammatory Th17 and immunosuppressive
Tregs is of crucial importance for homeostasis and overall immunity, one being the active
participant in generating inflammatory environment, and the latter being a suppressor
of further pathological processes, activation, proliferation, and effectors function [3,6].
Proliferative capacity and accumulation of inflammatory T cells can be suppressed by
dietary n-3 PUFAs intake, which leads to altered immune response through effects on lipid
mediators, cytokines, and immune cell abundance [7,8].
Lipid mediators, derived from essential n-3 or n-6 polyunsaturated fatty acids (PU-
FAs) through cyclooxygenase (COX) and lipoxygenase (LOX) enzymatic pathways [9,10],
and immune-cell-derived cytokines have significant effects on the innate and adaptive
immune system [11–13]. Pro-inflammatory prostaglandins (PGs) and leukotrienes (LTs)
are derivatives of long-chain n-6 PUFA-arachidonic acid (AA), while minimally inflamma-
tory oxylipins (PG, LT) and inflammation resolving resolvins (Rvs), protectins (PDs), and
maresins (MaRs) originate from long-chain n-3 PUFAs-α-linolenic (ALA), eicosapentaenoic
(EPA) and docosahexaenoic acid (DHA) [9]. AA derivatives play a crucial role in triggering
and maintaining the inflammation, while EPA and DHA derivatives have a role in termi-
nating the inflammatory response and blocking further cell recruitment while promoting
phagocytosis and tissue recovery [10,14].
Due to their involvement in inflammation and its resolution, the intake ratio between
n-3 and n-6 PUFAs is of great importance for homeostasis and overall well-being [15].
There has been a shift in Western diet towards increased dietary intake of n-6 PUFAs and
a devastating decline in n-3 PUFAs intake, with the ratio being 15–30:1, as opposed to
ideal 2–6:1 [15,16]. As a result, the balance between the pro- and the anti-inflammatory
derivatives that are synthesised from fatty acid precursors is disrupted, contributing to the
pathogenesis of CVD and cancer [15–18].
There has been growing clinical end epidemiological evidence of positive effects of
dietary n-3 PUFAs intake on the chronic inflammatory disorders, in terms of immunomod-
ulatory, anti-inflammatory, and cardioprotective effects [19–22]. Such beneficial impacts
were observed in human models, which include increased myokine irisin serum levels,
elevated gene expression of sirtuins, decreased TLR4 expression, reduced LDL-cholesterol
and hsCRP serum levels [19,23–25], while in animal models, previously reported effects
included decreased levels of pro-inflammatory, and increased levels of anti-inflammatory
cytokines, reduced placental oxidative stress and infarct size [26–29].
Food with biologically active properties (n-3 PUFA-enriched foods) can potentially
provide a sufficient tool for the protection from future chronic inflammation-mediated
diseases [21,30–32]. Recently, we have demonstrated that hen egg consumption signifi-
cantly increased systemic levels of anti-inflammatory cytokine IL-10—an effect that was
significantly more pronounced if the hen eggs were enriched with n-3 PUFAs [21]. In
addition, young healthy individuals who consumed a diet containing n-3 PUFA-enriched
hen eggs had lower levels of serum interferon gamma (INF-γ) after diet, suggesting posi-
tive effects of n-3 PUFA on systemic low-grade inflammation. Thus, the main objective of
the present study was to address the effects of n-3 PUFA-enriched hen eggs consumption
on the systemic levels of pro-/anti-inflammatory lipid mediators and the frequencies of
peripheral immune cells in young, healthy individuals without any underlying chronic
conditions. More specifically, we aimed to determine if the changes in the production of
lipid mediators originating from n-6 and n-3 PUFAs, induced by functional food intake,
have any effect on the peripheral T cell activation and differentiation status (Th17 and Treg).

2. Materials and Methods

2.1. Study Design and Participants
This was a randomised, double-blind, placebo-controlled study (part of ID NCT02720250
Omega-3 Fatty Acids Enriched Food and Microvascular Reactivity). The research was
carried out at the Department of Physiology and Immunology, Faculty of Medicine in
 2. Materials and Methods
2.1. Study Design and Participants
This was a randomised, double-blind, placebo-controlled study (part of ID
Nutrients 2021, 13, 1851 NCT02720250 Omega-3 Fatty Acids Enriched Food and Microvascular Reactivity). 3 of 20 The
research was carried out at the Department of Physiology and Immunology, Faculty of
Medicine in Osijek, Croatia. A total of 44 young and healthy volunteers were assessed for
eligibility. Two volunteers decided to decline participation in the study protocol, while
Osijek, Croatia. A total of 44 young and healthy volunteers were assessed for eligibility. Two
two participants who were enrolled in the study, failed to finish the study protocol due to
volunteers decided to decline participation in the study protocol, while two participants
personal
who werereasons. Ultimately,
enrolled 40failed
in the study, young healthy
to finish theadults of both due
study protocol sexes, aged between
to personal reasons.19–28
years old (23.840±young
Ultimately, 2.57 years old),
healthy participated
adults in this
of both sexes, agedstudy. Exclusion
between criteria
19–28 years oldfor
(23.8partici-
pants were
± 2.57 smoking,
years prior historyinofthis
old), participated hypertension, renal criteria
study. Exclusion or cerebrovascular impairments,
for participants were
coronary
smoking, artery
priordisease,
history ofdiabetes mellitus,
hypertension, renaland chronic inflammatory
or cerebrovascular disorders.
impairments, coronary Volun-
artery
teers whodisease,
met thediabetes mellitus,
inclusion and chronic
and exclusion inflammatory
criteria disorders.
were included in Volunteers
the study by who the re-
met the inclusion and exclusion criteria were included in the study
searcher and were awarded a label indicating the project, group of respondents by the researcher and
were awarded a label indicating the project, group of respondents (“healthy”), and ordinal
(“healthy”), and ordinal number. A CONSORT diagram is presented in Figure 1. A CON-
number. A CONSORT diagram is presented in Figure 1. A CONSORT checklist is presented
SORT checklist is presented as supplementary material (Supplementary Figure S1).
as supplementary material (Supplementary Figure S1).

Figure
Figure1.1.CONSORT 2010flow
CONSORT 2010 flowdiagram.
diagram.

TheThe studyprotocol
study protocollasted
lasted for
for three
threeweeks
weeksand included
and includedtwotwo
appointments. All study
appointments. All study
participants were instructed to eat three hard-boiled hen eggs (M commercial size) per
participants were instructed to eat three hard-boiled hen eggs (M commercial size) per
day for the duration of the protocol (three weeks). Participants were divided into two
daygroups:
for thecontrol
duration of (N
group the= protocol
21; W/M (three weeks).
= 10/11) Participants
consumed regular hen were
eggsdivided into two
(n-3 PUFAs
groups: control
content group (N
~249 mg/per = 21;
day), W/M
while n-3=PUFAs
10/11)group
consumed regular
(N = 19; W/M hen eggs
= 10/9) (n-3 PUFAs
consumed n-3 con-
tentPUFA-enriched
~249 mg/per day), while
hen eggs (n-3n-3 PUFAs
PUFAs group
content (N =mg/per
~1053 19; W/M = 10/9)
day). consumed
The dates n-3 of
of arrival PUFA-
the respondents were scheduled in advance by the researcher. Eggs were divided by an
unbiased associate according to the prearranged schedule of arrivals that did not contain
personal data but previously assigned labels. The simple randomisation procedure was
Nutrients 2021, 13, 1851 4 of 20

performed using a coin (letter-1 or head-2) to assign group affiliation by the unbiased
associate who added the number 1 or 2 in brackets to the previously mentioned label
for each individual subject, after the ordinal number, depending on which group it is:
1–a control group that consumed regular hen eggs; 2–n-3 PUFAs group that consumed
n-3 PUFA-enriched eggs. Labels indicating group affiliation were known only to the
associate assigning the intervention, while neither study participants nor the researcher
knew to which group they belonged throughout the duration of the study. This particular
associate did not participate in any of the performed analyses related to this specific
study population.
Enrichment of hen eggs was executed by an associate research group from the Faculty
of Agrobiotechnical Sciences Osijek, Josip Juraj Strossmayer University of Osijek, by replac-
ing soybean oil (5%) with a mixture of fish (1.5%) and linseed (3.5%) oil in feed mixtures
fed to laying hens. The fatty acid profile of the edible part of eggs used in the study was
previously elaborated by our research group [21]. In short, each enriched egg (edible part
~60 g) contained approximately 351 mg of n-3 PUFAs in total (~230 mg of ALA, ~15 mg
of EPA, ~105 mg of DHA). Participants consumed a total of 63 hard-boiled eggs during
the study.
Study participants kept a diet diary in the form of 24 h recalls during study protocol,
previously designed and published by our research group [33]. They were instructed to
follow their usual meal schedule and to avoid taking any supplements that could alter final
results, especially other n-3 PUFA supplements. At each appointment, the blood samples
were taken for peripheral blood mononuclear cell isolation and serum collection. Between
appointments, participants were contacted several times via E-mail and/or phone call to
assure compliance to study protocol.
The primary outcome of the study included a change in the frequency of T lymphocytes–
Tregs and Th17–after the dietary protocol, while secondary outcomes included quantifica-
tion of serum levels of lipid mediators originating from n-6 (LTB4, PGE2) and n-3 fatty acids
(LTB5, PGE3, RvE1), and pro- (IL17A, IL-23, IL-6) and anti-inflammatory cytokines (IL-10),
growth factor (TGF-β1) and chemokine (MCP-1) secretion following PMA–ionomycin
activation. Preliminary data were gathered from a total of 10 respondents after the same
dietary protocols as described above (N(Control) = 5; N (n-3 PUFA) = 5), considering
the primary outcome. The same simple randomisation procedure was performed and
given the results obtained, effect and sample size were calculated before the recruitment of
participants for the main study.
This study was conducted according to the guidelines laid down in the Declaration
of Helsinki, and all procedures involving research study participants were approved by
the Ethical Committee of the Faculty of Medicine, University of Osijek (CLASS: 602-04/20-
08/07; Reg. No.:2158-61-07-20-25). No selection bias was present in our study. All study
participants signed informed consent prior to the inclusion in the study, and there was no
compensation provided for their participation. Fresh eggs were delivered from the farm
(Marijančanka d.o.o.) to the laboratory once a week and distributed to participants entering
the protocol within 7 days.

2.2. Anthropometry and Laboratory Testing

Venous blood samples were taken after an overnight fast on the first and last day
of the protocol. Samples were analysed for full blood cell count, plasma electrolytes
(sodium, potassium, calcium), iron, transferrin, creatinine, urea, fasting blood glucose,
high-sensitivity C-reactive protein (hsCRP), and fasting lipid profile (total cholesterol,
low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides)
using standard laboratory methods and operating protocols at the Clinical Department of
Laboratory Diagnostics, University Hospital Osijek. All analyses were performed on Olym-
pus instrument using IVD certified reagents and protocols provided by the manufacturer.
Sodium, potassium, and calcium were measured by potentiometry; hsCRP, transferrin, and
ferritin (iron) were measured using immunoturbidimetric assays; blood cell count, haema-
Nutrients 2021, 13, 1851 5 of 20

tocrit levels, red cell indices (MCV, MCH, MCHC), RDW-CV, and MPV were evaluated
by impedance spectroscopy; other parameters were measured by spectrophotometry. The
lipid profile was measured directly.
Body mass index (BMI) was calculated according to the standard formula (BMI =
body mass/height in m2 ) using body mass (kg) and height (m) measures obtained at each
appointment by the researcher.

2.3. Peripheral Blood Mononuclear Cells (PBMCs)

2.3.1. Isolation from Whole Blood
Venous blood samples were collected in 10 mL vacutainer tubes containing EDTA
and processed within three hours of collection. Refrigerated reagents and buffers used in
isolation were warmed up to room temperature (RT, ~20–25 ◦ C) prior to isolation. Collected
whole blood was diluted with pre-prepared 1× phosphate-buffered saline (PBS) at 1:1
ratio and carefully layered on Ficoll-Paque® PLUS centrifugation media (GE Healthcare
Bio-Sciences AB, Uppsala, Sweden) without mixing the layers. Following, samples were
centrifuged for 25 min at 800 G with breaks off, at RT (Rotina 380, Hettich GmbH & Co.
KG, Tuttlingen, Germany). The layer containing mononuclear cells was collected and
rinsed twice with 1× PBS. Cell numbers were determined by staining the cells with 0.4%
Trypan blue solution (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and using a
Bürker-Türk counting chamber. (Accessed date 20 June 2019; Modification of protocol
available at https://www.cptp.inserm.fr/wp-content/uploads/2018/01/PBMC-isolation-
and-cryopreservation.pdf).

2.3.2. Cell Culture

PBMCs were cultured in RPMI-1640 media with L-glutamine (Sigma-Aldrich), supple-
mented with the addition of fetal bovine serum (10%) (FBS; Sigma-Aldrich) and penicillin–
streptomycin antibiotic (1%) (Capricorn Scientific GmbH, Ebsdorfergrund, Germany). Cell
suspensions were stored in 24-well plates and placed in an incubator (Shel Lab, CO2 Series,
Sheldon manufacturing Inc, Cornelius, OR, USA) under the following conditions: ~37 ◦ C,
5% CO2 , >80% humidity level for 24 h before any further proceedings.

2.3.3. Cryopreservation and Thawing

For the purpose of cryopreservation, dimethyl sulfoxide (DMSO; Supelco, Merck
KGaA, Darmstadt, Germany) and FBS were used at a 1:9 ratio. Additionally, for optimal
cell preservation, the cryovials were stacked in Mr. Frosty freezing container containing
isopropyl alcohol and placed in a −80 ◦ C freezer for at least 24 h.
Thawing of samples was carried out with FBS/antibiotics supplemented RPMI-1640
culture media preheated to ~37 ◦ C. Prior to adding the media, cryovials containing cells
were carefully dipped into a water bath (~37 ◦ C) for roughly 1 min and then transferred to
larger tubes. The preheated medium was pipetted onto the cells in a drip mode to avoid
osmotic shock and the samples were centrifuged. After two additional washing steps,
cells were resuspended in a fresh medium, transferred to 24-well plates, and kept in an
incubator for 24 h (~37 ◦ C, 5% CO2 , >80% humidity).

2.3.4. Cell Viability

Analyses to identify cell viability and exclude possible bias induced by nonspecific
staining of dead/dying cells in our samples included (a) staining cells with 0.4% Trypan
blue solution and counting live cells in the Bürker-Türk chamber under a light microscope
and (b) staining cells with fixable viability dye (FVD) eFluor™ 780 (eBioscienceTM , Invit-
rogen by Thermo Fisher Scientific, Waltham, MA, USA), which is detectable on a flow
cytometer upon excitation with 633 nm red laser. Samples included in the final analysis
and calculations had cell viability of ≥80%.
Nutrients 2021, 13, 1851 6 of 20

2.3.5. Magnetic Cell Sorting

After thawing the samples and adjusting the cell numbers to 1.2 × 107 , CD4+ T cells
were separated using negative magnetic selection via commercially available magnetic
beads (MagniSortTM Human CD4+ T cell, Enrichment kit; Invitrogen by Thermo Fisher
Scientific, Waltham, MA, USA), and following the protocol provided by the manufac-
turer (Accessed date 11 May 2020; Protocol available at https://assets.thermofisher.com/
TFS-Assets/LSG/manuals/8804-6811.pdf). Negatively selected cells were prepared for
the activation.

2.3.6. Activation of CD4+ T Lymphocytes

In order to activate CD4+ T cells and promote cytokine production, magnetically
sorted cells were shortly (4 h) stimulated by phorbol 12-myristate 13-acetate (PMA) and
ionomycin. CD4 T cell activation was carried out in 24-well plates (4 hrs, ~37 ◦ C, 5%
CO2 , >80% humidity level) with a commercially available cell stimulation cocktail (500×;
eBioscienceTM , Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) at a final
concentration of 2 µL/mL (Accessed date 12 May 2020; full protocol available at https:
//assets.thermofisher.com/TFS-Assets/LSG/manuals/00-4970.pdf).
Calcium Chloride (CaCl2 ) was added to the stimulation media to provoke a long-
lasting intracellular calcium signalling that would evoke a cellular response (5 µL/mL final
concentration).
In order to prevent cytokine secretion and allow assessment of IL-17 producing
cells by flow cytometry (intracellular IL-17 staining, detailed protocol given in Section 2.4),
Brefeldin A solution (1000×; eBioscienceTM , Invitrogen by Thermo Fisher Scientific, Waltham,
MA, USA) was used as an inhibitor of protein transport to Golgi apparatus with result-
ing accumulation of proteins in the endoplasmic reticulum (Accessed date 12 May 2020;
3 µL/mL final concentration; available at https://assets.thermofisher.com/TFS-Assets/
LSG/manuals/00-4506.pdf).
Cell stimulation cocktail, Brefeldin A and CaCl2 were added together and at the same
time to the cell suspension. After completion of four-hour incubation, 200 µL of 0.1 M
EDTA was added and incubated for 15 min at RT in order to stop the reaction.

2.4. Flow Cytometry

Frequencies of CD4+ Foxp3+ regulatory T lymphocytes and CD4+ IL-17A+ T helper lym-
phocytes among isolated peripheral blood mononuclear cells were determined by the flow
cytometry method. Sample preparation and staining protocols for intracellular antigens for
flow cytometry were modified versions of recommended protocols (Accessed date 13 May
2020; available at www.thermofisher.com). For intracellular staining, a Foxp3 transcription
factor staining buffer set was used (eBioscienceTM , Invitrogen by Thermo Fisher Scientific,
Waltham, MA, USA). In short, prior to cell surface staining, fixation/permeabilisation,
and intracellular/nuclear staining steps, dead cells were irreversibly labelled with previ-
ously mentioned FVD, and nonspecific antibody capturing by Fc receptors was blocked
by the addition of human Fc-blocking reagent (BD PharmigenTM , BD Biosciences, Becton,
Dickinson and Company, Franklin Lakes, NJ, USA). After incubation at RT, staining with
appropriate antibody mixture was carried out depending on the cell subset of interest.
Along with careful sample preparation and optimisation of staining protocols, single-stain,
fluorescence minus one (FMO), unstained and negative controls were included in our ex-
periments in order to reliably distinguish positive cells from background/negative staining
and nonspecific effects.
The fluorescence compensation matrix for multicolour flow cytometry analysis was
calculated using BDTM CompBeads Anti-Mouse Ig, κ/Negative Control Compensation
Particle Set (BD Biosciences, Becton, Dickinson and Company, Franklin Lakes, NJ, USA).
Measurements of stained samples were carried out by BD FACSCanto II cytometer (FAC-
SCanto II, Becton Dickinson, San Jose, CA, USA) equipped with blue Argon 488 nm and
Nutrients 2021, 13, 1851 7 of 20

Red HeNe 633 nm laser lines. Data analysis and visualisation were performed using the
FlowLogic software (Inivai Technologies, Mentone, Australia).

2.4.1. Regulatory T Lymphocytes (Tregs)

To assess the frequencies of Treg cells among PBMCs, the following mouse anti-
human antibodies mixture for the cell surface staining was used: CD3 FITC (clone: OKT3,
eBioscienceTM , Affymetrix by Thermo Fisher Scientific, CA, USA), CD4 PerCP-eFluorTM
710 (clone: SK3, eBioscienceTM ), CD127 PE-Cy7 (clone: eBioRDR5, eBioscienceTM ), CD25
APC (clone: BC96, eBioscienceTM ); while Foxp3 PE (clone: 235A/E7, eBioscienceTM )
antibody was used for intracellular staining of cells. The optimal antibody concentration
was determined by antibody titration experiment on 1 × 106 PBMCs and based on stain-
index calculations.

2.4.2. Helper T Lymphocytes (Th17)

Th17 lymphocytes’ rate among total peripheral blood CD4 lymphocytes were deter-
mined using PMA–ionomycin-activated CD4 cells and the following mouse anti-human
antibodies mixture: CD3 PerCP-eFluorTM 710 (clone: SK7, eBioscienceTM , Affymetrix by
Thermo Fisher Scientific, CA, USA), CD4 PE-Cy7 (clone: SK3, eBioscienceTM ), CD196 APC
(clone: R6H1, eBioscienceTM ) for cell surface antigens, and RORGt PE (clone: AFKJS-9,
eBioscienceTM ) and IL-17A FITC (clone: eBio64DEC17, eBioscienceTM ) for intracellular anti-
gens.

2.5. Luminex Assay

2.5.1. Collection of Supernatants from PMA–Ionomycin-Treated PBMC Cell Cultures
After thawing, the PBMCs were allowed to rest and recover overnight in culture
media (~37 ◦ C, 5% CO2 , >80% humidity level). Before proceeding to the next step, cells
were counted, and the number of cells was adjusted to 300,000 cells per 200 µL of stimu-
lation media for each sample. Stimulation media consisted of supplemented RPMI-1640
culture media, PMA–ionomycin. and CaCl2 at previously mentioned final concentrations
(Section 2.3). PBMC activation was carried out in 96-well plates (4 h, ~37 ◦ C, 5% CO2 , >80%
humidity level). Following the 4-h incubation period, the collected supernatant was stored
at −80◦ C until analysis.

2.5.2. Multiplex and Simplex Protein Quantitation of Pro- and Anti-Inflammatory

Cytokines, Transforming Growth Factor, and Chemokine Supernatant Concentrations
Concentrations of pro- and anti-inflammatory cytokines, including interleukin 17A
(IL-17A), interleukin 23 (IL-23), interleukin 6 (IL-6), interleukin 10 (IL-10); and transforming
growth factor-beta 1 (TGF-β1); and the levels of monocyte chemoattractant protein-1 (MCP-
1) chemokine were measured using the Invitrogen ProcartaPlex antibody-based, magnetic
bead reagent kits (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) and panels
for multiplex and simplex protein quantitation on the Luminex 200 instrument platform
(Luminex Corp., Austin, TX, USA). Measurements were performed in the Laboratory of
Molecular and HLA Diagnostics of the University Hospital Osijek (Osijek, Croatia). Data
were analysed by using ProcartaPlex Analyst free software (eBioscience, Affymetrix by
Thermo Fisher Scientific, Waltham, MA, USA) and are expressed as a concentration in
picograms per millilitre.

2.6. ELISA
Serum concentrations of leukotriene B4 (LTB4), leukotriene B5 (LTB5) (Cusabio, Hous-
ton, TX, USA), prostaglandin E2 (PGE2), prostaglandin E3 (PGE3) (MyBioSource, My-
BioSource Inc., San Diego, CA, USA), resolvin E1 (RvE1) (MyBioSource, MyBioSource Inc.,
CA, USA) were measured by commercially available enzyme-linked immunosorbent assay
(ELISA) kits on compact absorbance reader for 96-well microplates (BioRad PR 3100 TSC,
Bio-Rad Laboratories, CA, USA).
Nutrients 2021, 13, 1851 8 of 20

2.7. Statistical Analysis

Statistical analyses were performed using Microsoft Excel 2016 (Microsoft Office
365, Microsoft Corporation, Redmond, WA, USA), Graph Pad Prism v6.01 (GraphPad
Software, San Diego, CA, USA), and SigmaPlot v11 (Systat Software, Inc., Chicago, IL,
USA) software. Cohen’s d (∆/SD) effect size was determined according to the primary
outcome of the study—change in the frequency of T lymphocytes, Tregs, and Th17. GPower
v3.1.9.7 (Heinrich Heine University Düsseldorf, Düsseldorf, Germany) was used for sample
size calculation.
During optimisation of the protocol and design of the research, a pilot study was con-
ducted with a total of 10 respondents. Effect size (Cohen d, ∆/SD) required for a statistical
strength of 80% with bilateral α = 0.05, paired t-test, before and after the dietary protocol is
1.1 for the Treg lymphocyte population and requires a sample of at least 13 subjects per
group (GPower v3.1.9.7). For the estimated, expected arithmetic means of the frequency
of Foxp3+ Treg lymphocytes in the peripheral blood of healthy individuals before (16.8%)
and after the dietary protocol (9.2%), with corresponding standard deviations of 6.1% and
7.2%, the difference the arithmetic mean is 9% which corresponds to a biologically large
effect. The effect size required for a statistical strength of 80% with bilateral α = 0.05, paired
t-test, before and after the dietary protocol is 0.9 for the Th17 lymphocyte population
and requires a sample of a minimum of 19 subjects per group. For estimated, expected
arithmetic means of the frequency of IL-17A+ Th17 lymphocytes in the peripheral blood
of healthy individuals before (0.72%) and after the dietary protocol (0.49%), with corre-
sponding standard deviations of 0.29% and 0.19%, the difference of arithmetic means is
11%, which corresponds to a biologically large effect.
The normality of distributions was tested by Shapiro–Wilk test. Data are presented as
average ± standard deviation (SD). Student’s t-test and Mann–Whitney tests were used
for group comparisons, while Paired t-test and Wilcoxon rank-sum tests were used to
test the differences between the measurements within a group. Correlations between
paired datasets were determined by the Spearman rank test. Two-tailed p < 0.05 was
considered significant.

3. Results
3.1. Anthropometric and Biochemical Characteristics of Study Population
The general and biochemical characteristics of the individuals enrolled to participate
in this study are shown in Table 1. There were no significant differences in the age be-
tween the control (23.8 ± 2.79 years old) and the n-3 PUFAs group (23.8 ± 2.34 years
old). Both sexes were equally represented in the study groups. The average BMI of both
control (24.2 ± 3.01 kg/m2 ) and n-3 PUFAs group (22.72 ± 3.53 kg/m2 ) was within the
normal range according to the WHO criteria for European population weight classifica-
tion (18.5–24.9 kg/m2 ). Based on the medical history and the performed laboratory tests,
all participants were healthy. Their full blood count, serum electrolytes, fasting blood
glucose levels and hsCRP were within the normal range. Baseline total cholesterol and
LDL-cholesterol levels were found to be slightly elevated in the control group, compared
to the n-3 PUFAs group (p = 0.012 and p = 0.037, respectively), and above recommended
upper limits of the population, while all other biochemical parameters were within the
normal reference range for the general population. In a previous paper by Stupin et al.
(2020) [21] including the same study population and protocol, we reported that levels of
hsCRP were not significantly changed by consumption of regular or n-3 PUFA-enriched
eggs, compared to respective baseline measurements, nor was there a difference in hsCRP
between the groups. In addition, there were no significant differences in serum total choles-
terol, triglycerides, LDL cholesterol, and HDL cholesterol concentrations after regular or
n-3 PUFA-enriched eggs consumption, compared to respective baseline values. According
to the anthropometric, biochemical, and hemodynamic parameters, body composition and
body fluid status were all evaluated in the previously published paper by Stupin et al.
(2020.); our study groups were uniformly distributed [21].
Nutrients 2021, 13, 1851 9 of 20

Table 1. General and biochemical characteristics of study population.

n-3 PUFA Reference

Parameter Control Group p
Group Range
N (W/M) 21 (10/11) 19 (10/9) - -
Age (years) 23.8 ± 2.79 23.8 ± 2.34 0.954 -
BMI (kg/m2 ) 24.2 ± 3.01 22.72 ± 3.53 0.168 18.5–24.9
Urea (mmol/L) 5.27 ± 1.32 5.91 ± 1.25 0.183 2.8–8.3
Creatinine (µmol/L) 78.85 ± 16.68 85.67 ± 18.29 0.289 49–90
Sodium (mmol/L) 138.1 ± 2.36 137.92 ± 1.51 0.812 137–146
Potassium (mmol/L) 4.15 ± 0.25 4.24 ± 0.23 0.283 3.9–5.1
Calcium (mmol/L) 2.44 ± 0.06 2.41 ± 0.07 0.265 2.14–2.53
Iron (µmol/L) 17.55 ± 5.96 19.34 ± 6.09 0.419 8.0–30.0
Transferrin (g/L) 2.87 ± 0.49 2.78 ± 0.39 0.62 2.00–3.60
Fasting blood glucose (mmol/L) 4.82 ± 0.57 4.64 ± 0.82 0.474 4.2–6.0
hsCRP (mg/L) 1.85 ± 2.41 1.53 ± 1.36 0.672 <5.00
0.012
Cholesterol (mmol/L) 5.26 ± 0.96 4.39 ± 0.74 <5.00
*
Triglycerides (mmol/L) 1.09 ± 0.48 1.25 ± 1.08 0.573 <1.70
HDL cholesterol (mmol/L) 1.61 ± 0.37 1.38 ± 0.29 0.071 >1.20
0.037
LDL cholesterol (mmol/L) 3.27 ± 0.81 2.71 ± 0.49 <3.00
*
Leukocytes (×10E9/L) 6.17 ± 1.38 6.12 ± 1.44 0.918 4.4–11.6
Platelets (×10E9/L) 256 ± 65.97 228.83 ± 36.82 0.202 178–420
Erythrocytes (×10E12/L) 4.77 ± 0.33 4.72 ± 0.39 0.692 4.07–5.42
Haemoglobin (g/L) 140.2 ± 10.99 142.75 ± 12.05 0.544 118–149
Haematocrit 0.41 ± 0.03 0.41 ± 0.03 0.702 0.354–0.450
MCV (fL) 86.11 ± 3.73 88.7 ± 3.15 0.054 76.5–92.1
MCH (pg) 29.45 ± 1.49 30.23 ± 1.12 0.126 24.3–31.5
MCHC (g/L) 341.85 ± 5.58 340.92 ± 6.36 0.667 304–346
RDW-CV (%) 13.8 ± 0.92 14.08 ± 0.51 0.35 9.0–15.0
MPV (fL) 10.43 ± 0.49 10.73 ± 0.58 0.129 7.0–10.4
Results are expressed as average ± standard deviation (SD). N—number of participants; W—women; M—men;
BMI—body mass index; hsCRP—high-sensitivity C-reactive protein; HDL—high-density lipoprotein; LDL—
low-density lipoprotein; MCV—mean corpuscular volume; MCH—mean corpuscular haemoglobin; MCH—
mean corpuscular haemoglobin concentration; RDW-CV—red cell distribution width; MPV—mean platelet
volume. Student’s t-test; significance level p < 0.05 * control group vs. n-3 PUFAs group. Reference range—
general population.

3.2. n-3 PUFA Supplementation Changes the Ratio of Pro- and Anti-Inflammatory Lipid Mediators
Originating from n-6 (AA) and n-3 (EPA) Fatty Acids
Serum concentrations of pro-inflammatory eicosanoids (LTB4 and PGE2) from AA,
and inflammation resolving oxylipins (LTB5 and PGE3) and resolvins (RvE1) from EPA
and DHA before and after the dietary protocols are shown in Figure 2.
LTB4 and PGE3 serum levels were significantly increased in the control group af-
ter three-week consumption of regular hen eggs (p = 0.021 and p = 0.014, respectively,
Figure 2B,C), while their levels remained unchanged in the n-3 PUFAs group. Average
serum concentrations of LTB5 at the end of the dietary protocol were significantly increased
in both groups, compared to their respective baseline levels (p < 0.0001 and p = 0.012,
respectively; Figure 2A). Serum level of RvE1 was significantly increased in the n-3 PU-
FAs group after the three-week consumption of n-3 PUFA-enriched hen eggs (p = 0.013;
Figure 2E). Serum concentrations of PGE2 were not significantly affected by any of the
dietary protocols (Figure 2D). To address the proportions of pro-inflammatory and anti-
inflammatory lipid mediators, prostaglandin E2/E3 and leukotriene B4/B5 ratios were
calculated and compared across the measurements. These results showed a significant
decrease in prostaglandin E2/E3 ratio following n-3 PUFA dietary protocol (p = 0.014;
Figure 2F), while the leukotriene B4/B5 ratio remained unchanged in both groups.
 after the three-week consumption of n-3 PUFA-enriched hen eggs (p = 0.013; Figure 2E).
Serum concentrations of PGE2 were not significantly affected by any of the dietary proto-
cols (Figure 2D). To address the proportions of pro-inflammatory and anti-inflammatory
lipid mediators, prostaglandin E2/E3 and leukotriene B4/B5 ratios were calculated and
compared across the measurements. These results showed a significant decrease in pros-
Nutrients 2021, 13, 1851 10 of 20
taglandin E2/E3 ratio following n-3 PUFA dietary protocol (p = 0.014; Figure 2F), while the
leukotriene B4/B5 ratio remained unchanged in both groups.

Figure 2. Effects of regular (control group) and n-3 polyunsaturated acid (PUFA)-enriched hen egg (n-3 PUFA group)
Figure 2. Effects of regular (control group) and n-3 polyunsaturated acid (PUFA)-enriched hen egg (n-3 PUFA group)
consumption on the serum concentrations of pro-inflammatory leukotriene B4 (B) and prostaglandin E2 (D); inflammation
consumption on the serum concentrations of pro-inflammatory leukotriene B4 (B) and prostaglandin E2 (D); inflammation
resolving
resolvingleukotriene
leukotriene B5B5
(A), prostaglandin
(A), prostaglandinE3 E3
(C)(C)
andand
resolvin E (E)E lipid
resolvin mediators
(E) lipid originating
mediators n-6 (arachidonic
from from
originating acid;
n-6 (arachidonic
AA)
acid;and
AA) n-3and
(eicosapentaenoic acid; EPA)
n-3 (eicosapentaenoic acid;fatty
EPA)acids.
fattyThe ratio
acids. between
The prostaglandin
ratio between E2 and prostaglandin
prostaglandin E3 is shown
E2 and prostaglandin E3 is
atshown
Panel at
(F). PUFA—polyunsaturated
Panel fatty acid;
(F). PUFA—polyunsaturated LTB4
fatty acid;– leukotriene B4; LTB5—leukotriene
LTB4 – leukotriene B5; PGE2
B4; LTB5—leukotriene B5;- PGE2
prostaglandin E2;
- prostaglan-
din E2; PGE3—prostaglandin
PGE3—prostaglandin E3; RvE1—resolvin
E3; RvE1—resolvin E1. PairedE1. Paired
t-test; t-test; significance
significance level
level p < 0.05; p < protocol
before 0.05; before protocol
vs. after vs. after
protocol.
protocol.

3.3. PBMC-Derived Cytokines Following PMA–Ionomycin Activation

Concentrations of anti-/pro-inflammatory cytokines (IL-10, IL-6, IL-23, IL-17A, and
TGF-β1) and MCP-1 chemokine in both control and experimental group, before and
after dietary protocols were determined in supernatants from PBMC cell cultures upon
PMA/ionomycin stimulation (Figure 3).
TGF-β1 production by peripheral blood mononuclear cells following n-3 PUFA dietary
protocol was significantly increased (p = 0.026; Figure 3A), while IL-6 production was
significantly decreased (p = 0.05; Figure 3D), compared to the respective baseline levels. In
addition, end-point TGF-β1 levels were significantly lower in the control group, compared
with the end-point levels measured in the n-3 PUFAs group (p = 0.004; Figure 3A). Target
cytokine and chemokine production by PBMC was unaffected by the consumption of
regular hen eggs (control group, Figure 3A–E). Furthermore, the intergroup analysis
revealed significant differences in baseline and end-point IL-17A production, namely, in
the control group, levels of IL-17 secreted by PBMC upon PMA/ionomycin stimulation
were significantly higher both prior and after consumption of regular hen eggs, compared
to n-3 PUFAs group (p < 0.001 and p = 0.001, respectively; Figure 3C).
 pared with the end-point levels measured in the n-3 PUFAs group (p = 0.004; Figure 3A).
Target cytokine and chemokine production by PBMC was unaffected by the consumption
of regular hen eggs (control group, Figure 3A–E). Furthermore, the intergroup analysis
revealed significant differences in baseline and end-point IL-17A production, namely, in
the control group, levels of IL-17 secreted by PBMC upon PMA/ionomycin stimulation
Nutrients 2021, 13, 1851 11 of 20
were significantly higher both prior and after consumption of regular hen eggs, compared
to n-3 PUFAs group (p < 0.001 and p = 0.001, respectively; Figure 3C).

Figure 3. Levels of pro-/anti-inflammatory cytokines and chemokines secreted by PBMCs upon PMA/ionomycin stim-
Figure 3. Levels of pro-/anti-inflammatory cytokines and chemokines secreted by PBMCs upon PMA/ionomycin stimula-
ulation in young individuals following regular (control Group) or n-3 polyunsaturated acid (PUFA)-enriched hen egg
tion in young individuals following regular (control Group) or n-3 polyunsaturated acid (PUFA)-enriched hen egg (n-3
(n-3
PUFAPUFA group)
group) consumption.
consumption. ThereThere
was awas a significant
significant increase
increase in TGFβ-1
in TGFβ-1 level
level in n-3 in n-3 PUFA
PUFA group group (A), no
(A), while while no sig-
significant
nificant changes were found for IL-10 (B), IL-17A (C), IL-6 (D) or MCP-1 (E). PBMC—peripheral blood
changes were found for IL-10 (B), IL-17A (C), IL-6 (D) or MCP-1 (E). PBMC—peripheral blood mononuclear cells; TGFβ- mononuclear
cells; TGFβ-1—Transforming
1—Transforming Growth FactorGrowth Factor
Beta-1; Beta-1; IL-10—Interleukin
IL-10—Interleukin 10; IL-17A—Interleukin
10; IL-17A—Interleukin 17A; IL-6—Interleukin
17A; IL-6—Interleukin 6; MCP-1— 6;
MCP-1—Monocyte Chemoattractant
Monocyte Chemoattractant Protein-1.Protein-1. Paired
Paired t-test; t-test; significance
significance level p < level p < 0.05;
0.05; before before protocol
protocol vs. after protocol.
vs. after protocol.

3.4. Frequencies/Abundance of Peripheral Blood Treg and Th17 Lymphocytes Are Reduced
Following Dietary Protocols
Both dietary protocols resulted in significant decrease of CD25/Foxp3-expressing
peripheral blood lymphocytes within CD4+ CD127+ subpopulation (p < 0.001; Figure 4A,B).
The same differences were observed when frequencies of these cells (CD25+ Foxp3+ ) were
compared within the total peripheral T helper pool (p < 0.001 and p = 0.002 for the control
group and n-3 PUFAs group, respectively; Figure 4A,B). The observed decrease was 1.5-fold
in the control group and 1.6-fold in the n-3 PUFAs group. Additional analysis of Foxp3
expression in T helper cells showed that the observed differences were due to the significant
reduction of CD4+ CD25+ Foxp3high subpopulation corresponding to ‘real’ regulatory T
cells (nTreg) in both groups (Figure 4C,D), while the observed increase in recently activated
CD4+ CD25+ Foxp3int T cell frequencies represents a relative increase since their frequencies
within the total T cell pool remained unchanged (Figure 4Civ). There were no significant
differences in these subpopulations of cells between the groups, neither prior to entering
the dietary protocols nor at the end of the protocols. An additional finding of this study
was that the rates of recently activated T cells were positively associated, while the % of
nTreg was inversely related to serum IL-22 levels in the n-3 PUFAs group (Figure 4D).
 Nutrients 2021, 13, x FOR PEER REVIEW 13 of 20

no association to ‘real’ Th17 cells. Leukotriene B5 serum concentrations negatively corre-

Nutrients 2021, 13, 1851 12 of 20
lated with concentrations of PBMC-derived IL-17A (r = −0.743, p = 0.029). HDL-cholesterol
levels negatively correlated with hsCRP (r = −0.408, p = 0.042).

.
Figure 4. Effects of regular (control group) and n-3 polyunsaturated acid (PUFA)-enriched hen egg (n-3 PUFAs group)
Figure 4. Effects of regular (control group) and n-3 polyunsaturated acid (PUFA)-enriched hen egg (n-3 PUFAs group)
consumption
consumption on on
thethe
frequency of peripheral
frequency regulatory
of peripheral T cells
regulatory (Treg)
T cells in healthy
(Treg) young
in healthy individuals.
young (A) shows
individuals. representative
(A) shows representa-
dottive
plots illustrating gating strategy, including exclusion of doublets using forward scatter area (FSC-A) versus
dot plots illustrating gating strategy, including exclusion of doublets using forward scatter area (FSC-A) versus forwardfor-
scatter width (FSC-W) analysis (A-i), gating on live cells negative for amine-reactive fixable viability dye (A-ii),
ward scatter width (FSC-W) analysis (A-i), gating on live cells negative for amine-reactive fixable viability dye (A-ii),lymphocytes
(A-iii) and CD3+ T cells (A-iv). First, total CD25 and Foxp3 expressing T cells among CD4+ CD127low population were
analysed (B). B-i shows representative gating strategy, while the relative frequencies are presented as box-and-whisker plots
at B-ii. Next, we have analysed CD25+ CD127low subpopulation (C-ii) of T helper lymphocytes (CD3+ CD4+ ) (C-i) for the
Nutrients 2021, 13, 1851 13 of 20

expression of Foxp3 transcription factor (C-iii). C-i/ii/iii shows representative gating strategy, while the relative fre-
quencies of CD25+ Foxp3int and CD25+ Foxp3high are presented as box-and-whisker plots at (C-iv) and C-v, respectively.
Based on the Foxp3 expression (B,C), two T cell subpopulations were identified within CD4+ CD127− CD25+ T cell pool:
CD4+ CD127- CD25+ Foxp3int recently activated T helper cells (Ci–iv); and CD4+ CD127− CD25+ Foxp3high subpopulation
corresponding to regulatory T cells (Ci–iii, v). (D) shows differential correlation of Foxp3-expressing subpopulations
with TGFβ-1 (secreted upon PBMC stimulation) and serum levels of IL-22. PUFA—polyunsaturated fatty acid; TGFβ-1—
Transforming Growth Factor Beta-1; PBMC—peripheral blood mononuclear cells; IL-22 - Interleukin 22. Paired t-test, before
protocol vs. after protocol; p < 0.05 was considered significant; r—Spearman correlation coefficient.

In the present study, a significant reduction in the frequency of total IL-17 secreting
peripheral T helper cells was observed at the end of the protocol in the control group
(p = 0.008; Figure 5Bi,ii). These cells were further immunephenotyped and, based on their
CCR6 expression, subdivided to Th17 (CCR6+ IL-17+ ) and non-Th17 (CCR6- IL-17+ ) T helper
cells, with the latter corresponding mostly to IL-17-secreting Th1 and Th2 T helper cells.
This further revealed that the frequencies of Th17 cells were significantly reduced at the
end of both dietary protocols (p = 0.009 and p = 0.008 in the case of the control group and
n-3 PUFAs group, respectively; Figure 5Ci,ii). Interestingly, in the case of CCR6- IL-17+ T
cells (non-Th17 cells), the control group had significantly reduced frequency of these cells
(p = 0.033, Figure 5Ciii), while the subjects from the n-3 PUFAs group had significantly
increased frequency of the same T cell subpopulation (p < 0.001, Figure 5Ci).

3.5. Correlation Analysis

Correlation analysis was performed to assess relationships between biochemical
parameters (hsCRP, fasting lipid profile), serum concentrations of eicosanoids and resolvins,
PBMC-derived cytokines and chemokines, and peripheral Treg and Th17 lymphocyte
frequencies.
In the control group, rates of peripheral blood CD25/Foxp3-expressing lymphocytes
were positively associated with the rates of peripheral blood IL-17 producing CD4 T cell
subset (r = 0.326; p = 0.035) and inversely associated with the serum fasting cholesterol
levels (r = −0.332; p = 0.036) and BMI (r = −0.441; p = 0.0036). However, after further iden-
tification of nTreg (CD25+ Foxp3high ) and recently activated T cells (CD25+ Foxp3int ) among
CD25/Foxp3-expresing T helper cells, associations with CD4+ IL-17+ T cell pool were lost.
Interestingly, the BMI status was still inversely related to nTreg (r = −0.535; p < 0.0001) but
positively associated to the rates of recently activated T helper cells (r = 0.399; p = 0.009).
There was a positive correlation between prostaglandin E2 and E3 serum concentrations
(r = 0.819; p = 0.0002), while there was a significant negative correlation found between
hsCRP level and prostaglandin E2/E3 ratio (r = −0.438; p = 0.006).
In the n-3 PUFAs group, peripheral nTreg lymphocytes were negatively associated
with non-Th17 IL-17A secreting T helper cells (CCR6- IL-17+ ; r = −0.613, p < 0.0001) and
PBMC-derived TGFβ-1 (r = −0.616, p = 0.002), while recently activated T helper cells were
positively associated with CCR6- IL-17+ (r = −0.591, p = 0.0003) and PBMC-derived TGFβ-1
(r = −0.522, p = 0.012). Interestingly, rates of peripheral non-Th17 lymphocytes were
positively associated with PBMC-derived IL-17A levels (r = 0.456, p = 0.032), while there
was no association to ‘real’ Th17 cells. Leukotriene B5 serum concentrations negatively
correlated with concentrations of PBMC-derived IL-17A (r = −0.743, p = 0.029). HDL-
cholesterol levels negatively correlated with hsCRP (r = −0.408, p = 0.042).
 tive frequencies of CD25+Foxp3int and CD25+Foxp3high are presented as box-and-whisker plots at (C-iv) and C-v, respec-
tively. Based on the Foxp3 expression (B and C), two T cell subpopulations were identified within CD4+CD127−CD25+ T
cell pool: CD4+CD127-CD25+Foxp3int recently activated T helper cells (Ci-iv); and CD4+CD127−CD25+Foxp3high subpopula-
tion corresponding to regulatory T cells (Ci–iii, v). (D) shows differential correlation of Foxp3-expressing subpopulations
with TGFβ-1 (secreted upon PBMC stimulation) and serum levels of IL-22. PUFA—polyunsaturated fatty acid; TGFβ-1—
Nutrients 2021, 13, 1851 14 of 20
Transforming Growth Factor Beta-1; PBMC—peripheral blood mononuclear cells; IL-22 - Interleukin 22. Paired t-test, be-
fore protocol vs. after protocol; p < 0.05 was considered significant; r—Spearman correlation coefficient.

Figure
Figure Effects
5. 5. of of
Effects regular
regular(control
(controlgroup)
group)and n-3n-3
and polyunsaturated
polyunsaturated acid (PUFA)-enriched
acid (PUFA)-enriched hen egg
hen (n-3
egg (n-3PUFAs
PUFAs group)
group)
consumption on the representation of peripheral T helper cell subpopulations in healthy
consumption on the representation of peripheral T helper cell subpopulations in healthy young individuals: (A)young individuals: (A) shows
shows
representative
representative dotdotplots illustrating
plots illustrating gating strategy,
gating strategy, including
includingexclusion
exclusionof of
doublets
doubletsusing
usingforward
forwardscatter
scatter area
area(FSC-A)
(FSC-A)
versus
versus forward
forward scatter
scatter width
width (FSC-W)
(FSC-W) analysis
analysis (A-i),
(A-i), gating
gating onon live
live cells
cells negative
negative forfor amine-reactive
amine-reactive fixable
fixable viability
viability dyedye
(A-ii), lymphocytes (A-iii), CD3
+ + T cells (A-iv), and CD3 + +CD4
+ + T cells (A-v). T helper cells were subsequently analysed for
(A-ii), lymphocytes (A-iii), CD3 T cells (A-iv), and CD3 CD4 T cells (A-v). T helper cells were subsequently analysed for
IL-17A
IL-17A (B)(B)
andand CD196/CCR6
CD196/CCR6 expression
expression (C).(C). First;
First; all IL-17
all IL-17 secreting
secreting T helper
T helper cellscells
werewere analysed
analysed (B) where
(B) where the on
the gate gate
IL-on
+IL-17+ T cells was defined using fluorescence minus one (FMO) control (B-i) and the relative frequencies + of CD4
+
+IL-17+ T
17 T cells was defined using fluorescence minus one (FMO) control (B-i) and the relative frequencies of CD4 IL-17 T helper
helper cells are presented as box-and-whisker plots at (B-ii). The population of IL-17 secreting T helper cells was further
cells are presented as box-and-whisker plots at (B-ii). The population of IL-17 secreting T helper+ cells was further analyse s
analyse s for CD196/CCR6 expression (C-i), hence two subpopulations were identified—CD4 + +
CD196
+
+IL-17 + corresponding
fortoCD196/CCR6 expression
Th17 cells (C-ii) and CD4 (C-i),
+CD196hence two+ non-Th17
-IL-17 subpopulations were identified—CD4
cells accounting CD196subpopulations
for other T helper IL-17 corresponding
with thetocapacity
Th17
cells (C-ii) and CD4+ CD196- IL-17+ non-Th17 cells accounting for other T helper subpopulations with the capacity to secrete
IL-17 (C-iii). There was a significant decrease in Th17 cell frequency in both groups following dietary protocol. Frequency of
non-Th17 cells was significantly reduced in control group, while the same T cell subpopulation was increased in the n-3
PUFAs group after dietary protocol. Paired t-test, before protocol vs. after protocol; p < 0.05 was considered significant.

4. Discussion
Beneficial effects of n-3 PUFA supplementation are well documented in the literature,
but these studies usually involved n-3 or n-6 PUFAs addition to various cell cultures [9,34,35]
or, in the case of human studies, oil capsules were mostly given to patients with certain
comorbidities [36–38]. In the present study, we aimed to determine the effects of n-3 PUFA
Nutrients 2021, 13, 1851 15 of 20

supplementation, i.e., ALA, EPA, and DHA, through functional foodstuffs, on pro- and
anti-inflammatory parameters in young healthy individuals. We previously established
that this particular study population normally consumes extremely low amounts of n-3
PUFA-rich food such as fish and nuts [33]; therefore, the main effect of n-3 PUFAs in the
present study came from the consumption of n-3 PUFA-enriched hen eggs. Assessment of
serum fatty acid profile before and after finishing respective dietary protocols confirmed
excellent compliance and significant changes to the fatty acid composition and n-6/n-3
PUFAs ratio [21]. The salient findings of the present study are differential effects of the
regular hen egg and n-3 PUFA-enriched hen egg consumption on the serum levels of
lipid mediators, representation of peripheral T helper cell subsets (recently activated T
helper cells, nTreg, Th17, and non-Th17 IL-17A secreting T helper lymphocytes), and
their functional capacity for cytokine secretion. Both diets significantly altered systemic
levels of pro-inflammatory, and inflammation resolving lipid mediators; however, only
the n-3 PUFAs group showed a significant shift towards anti-inflammatory prostanoids
and increased levels of pro-resolvins. Both study groups showed reduced frequencies of
peripheral nTreg lymphocytes and decreased rates of peripheral Th17 cells. Their functional
capacity for cytokine secretion was significantly altered only in the n-3 PUFAs group in
terms of increased TGFβ-1 and reduced IL-6 secretion.
Inhibitory effects of supplemental ALA and DHA on the COX pathway were previ-
ously documented in human umbilical vein endothelial cells (HUVECs) [9] and bovine
aortic endothelial cells (BAECs) [34,35]. In addition, Araujo et al. (2019) [9] failed to
find a significant association between increased EPA administration and production of
PGE3 in HUVECs, suggesting ALA/DHA-specific effects on the COX pathway. A possible
explanation was given by Malkowski et al. (2001) [39], who described decreased flexi-
bility of EPA when bound to COX activity site due to an additional double bond which
results in low oxygenation and enzymatic conversion to PGE3. This could also explain
the results of the present study in which we were not able to prove significant effects of
n-3 PUFAs supplementation through functional food on systemic levels of both pro- and
anti-inflammatory (PGE2 and PGE3, respectively) lipid metabolites derived via the COX
pathway. Even though there were no significant changes observed in PGE2 and PGE3
serum concentrations individually, there was a significant decrease in endpoint PGE2/E3
serum concentration ratio in the n-3 PUFAs group, compared to baseline ratio, which
indicates a slight shift in favor of anti-inflammatory metabolites.
Further finding of this study were significantly increased serum concentrations of
inflammation resolving five-series of leukotrienes/LTB5 in both groups at the end of dietary
protocols and increased levels of pro-inflammatory four-series of leukotrienes/LTB4 in
the control group which indicates that the activity of the LOX pathway is preferred over
the production of COX pathway metabolites from EPA. It has been previously shown that
supplemental DHA increases the production of LOX pathway metabolites and resolvins
in HUVECs [9]. This information and the fact that DHA can be retro-converted to EPA
following supplementation [40,41] support our finding of increased production of RvE1
in the n-3 PUFAs group following diet protocol. Both E- and D-series resolvins have a
crucial role in response to acute inflammation [42], especially in allergic response and
asthma [43,44], and neuroinflammation [14,45], and exhibit therapeutic potential for the
treatment of inflammatory bowel disease [46].
Examined lipid mediators and cytokines (secreted predominantly by T cells) act syner-
gistically to constrain inflammation and to promote resolution after pathogen elimination,
predominantly through refining T cell functions [11–13,43]. Cytokines act as signalling
molecules and humoral regulators of cell activation, differentiation, proliferation, and cy-
tokine production, including chemokines, interleukins (IL), growth factors, and interferons
(IF) [12,13,47]. T lymphocytes manage cell immunity through activation of phagocytes
and by promoting the destruction of pathogens and infected cells while also producing
cytokines with specific effects on the other immune cells [3,11]. Cytokine secretion by
Nutrients 2021, 13, 1851 16 of 20

T lymphocytes was significantly altered by n-3 PUFAs in our study towards reduced
inflammation, as further described.
Haworth et al. (2008) [43] reported that increased RvE1 decreases IL-17A concentration
by ~70% in an animal model. This effect, however, could not be observed in our study
population. As elaborated in the Results Section, in our cohort of healthy young adults, a
significant difference was observed regarding IL-17A levels in the control group, both prior
and after the protocol, when compared to the n-3 PUFAs group. Therefore, we performed
series of correlation analyses between the IL-17A levels and anthropometric/biochemical
parameters which could not explain the initial differences in IL-17A production between the
groups. Ongoing acute infection or underlying immune-mediated inflammatory disorders
were eliminated based on the medical records and blood tests. However, a significant
positive association was observed between RvE1 and systemic levels of IL-10, IL-22, IL-6,
and IL-9 only in the n-3 PUFAs group. In addition, the functional capacity of lymphocytes
to produce IL-17A was inversely related to systemic levels of pro-resolving LTB5 in the n-3
PUFAs group, which is, alongside RvE1, also an EPA-derived mediator. Furthermore, the
inhibitory effect of RvE1 on IL-6 production was previously reported in human neutrophil
cell lines [48], and IL-6 was another cytokine measured in our study, namely, IL-6 secretion
by lymphocytes upon PMA/ionomycin activation was significantly decreased in n-3 PUFAs
group at the end of the dietary protocol.
Above mentioned pro-inflammatory cytokines, alongside IL-23, have a detrimental
role in the differentiation and survival of Th17 cells as well as in mediating Th17 effector
functions. In addition, previous research suggests that they can be downregulated by
increased RvE1 production [43], which we have partly confirmed by the observation that
IL-6 production by peripheral lymphocytes was reduced, in parallel with increased systemic
levels of RvE1 in the n-3 PUFAs group. In addition, there was decreased prevalence of
peripheral Th17 lymphocytes following hen eggs consumption, independently of their n-3
PUFA enrichment.
TGFβ-1 and IL-10 cytokines have a central role in maintaining the immune balance by
limiting immune reactions and promoting additional inducible regulatory T cell differentia-
tion (iTreg) [49], thus preventing uncontrolled inflammation and/or autoimmunity [50–53].
Rosa et al. (2012) [54] demonstrated that tissue levels of TGFβ-1 were increased in rats fol-
lowing EPA and DHA administration through fish oil. Similarly, increased TGF-β1 mRNA
and protein secretion in colonic cell lines in response to commensal bacteria Lactobacillus
gasseri was enhanced by pre-treatment with EPA [55]. This is in the line with present finding
of enhanced capacity of activated PBMCs for TGFβ-1 secretion following consumption
of n-3 PUFA-enriched eggs. Interestingly, levels of secreted TGFβ-1 negatively correlated
with the frequency of peripheral nTreg cell population in the n-3 PUFAs group. Even
though TGFβ-1 exhibits both anti- and pro-inflammatory properties [56,57], the latter is
manifested only in combination with IL-6 (promotion of Th17 differentiation) [58] which
was decreased in the n-3 PUFAs group.
In healthy individuals, nTreg cells represent around 2–10% of the total T helper cells
pool, with slightly lower frequencies found in peripheral blood [59,60]. Treg cells have
shown noticeable therapeutic potential in terms of their expansion to control autoimmune
and inflammatory disorders or, at the other side of the therapeutic spectra, their depletion to
promote T effector cell function and eliminate cancer [59,61]. However, most recent studies
reported their substantial phenotypic and functional variability beyond sole immunosup-
pression [62–66]. Decreased rates of peripheral Treg cells following n-3 PUFA-enriched
functional food consumption in our study can be explained by the previously reported
inhibitory effect of dietary DHA on both migratory and suppressive Treg cells functions
which was proven as dose dependent in vitro and in vivo (100 µmol/L) [61,67]. Interest-
ingly, a diet rich in n-3 PUFAs upregulates expression of Treg cell markers, TGFβ-1, and
Foxp3 [61,67], which was confirmed by our results, and explains the negative correlation
between TGFβ-1 supernatant concentration and Tregs.
Nutrients 2021, 13, 1851 17 of 20

5. Conclusions
The results of the present study have demonstrated the important role of n-3 PUFAs on
immunoregulation when consumed in the form of functional foods. This is the first study
of its kind to determine that n-3 PUFAs can modify key mediators of inflammation and
alter the frequency of particular lymphocyte subpopulations in human subjects through
modified foodstuff. Such a set of achieved conditions can lead to a shift towards systemic
inflammation resolving environment in the population of young and healthy individuals.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10

.3390/nu13061851/s1, Figure S1: CONSORT 2010 checklist of information to include when reporting
a randomized trial.
Author Contributions: Conceptualisation, N.K., A.M., M.M. and I.D.; data curation, N.K. and
M.M.; formal analysis, N.K. and M.M.; funding acquisition, I.D.; investigation, N.K., A.M. and
M.M.; methodology, N.K., A.M., P.Š., Z.M. and M.M.; project administration, N.K., M.M. and I.D.;
resources, I.D.; software, N.K., A.M. and M.M.; supervision, M.M. and I.D.; validation, M.M. and
I.D.; visualisation, N.K., M.M. and I.D.; writing—original draft preparation, N.K., A.M., P.Š., Z.M.,
M.M. and I.D.; writing—review and editing, N.K., A.M., P.Š., Z.M., M.M. and I.D. All authors have
read and agreed to the published version of the manuscript.
Funding: The study was funded by European Structural and Investment Funds to Science Centre of
Excellence for Personalised Health Care, the Josip Juraj Strossmayer University of Osijek, Scientific
Unit for Research, Production and Medical Testing of Functional Food, # KK.01.1.1.01.0010.
Institutional Review Board Statement: This study was conducted according to the guidelines laid
down in the Declaration of Helsinki and all procedures involving research study participants were
approved by the Ethical Committee of the Faculty of Medicine, University of Osijek (CLASS: 602-
04/20-08/07; Reg. No.:2158-61-07-20-25).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: Special thanks to all the participants for their valuable contribution to the study.
Conflicts of Interest: The 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.

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