1.   Abbreviations:

LF                           :               Lactoferrin

TNF-α                     :               Tumor Necrosis Factor Alpha

LPS                        :               Lipopolysaccharide

IL-10                      :               Interleukin-10

IL-12                      :               Interleukin-12

hLF                        :               Human Lactoferrin

bLF                        :               Bovine Lactoferrin

LF10                      :               Lactoferrin 10 µg/mL

LF40                      :               Lactoferrin 40 µg/mL

PC                         :               Positive Control

FBS                       :               Fetal Bovine Serum

MTT                       :               3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

GAPDH                 :               Human glyceraldehyde-3-phosphate dehydrogenase

SD                         :               Standard Deviation

SEM                      :               Standard Error from Mean

ROS                      :               Reactive Oxygen Species

2. Introduction

Lactoferrin (LF), is an 80 kDaglycoprotein belonging to the transferrin family of non-heme iron binding proteins together with transferrin [1,2,3], ovotransferrin [4], melanotransferrin [5] and a recently identified carbonic anhydrase inhibitor [6].The main role of these proteins is to control the levels of free iron in biological fluids. LF was discovered for first time from bovine milk by Sorensen and Sorensens in 1939 [7] and then purified by three independent laboratories in 1960 [8-10].

It consists of a single polypeptide chain of 703 amino acids, folded in two globular lobes, C (carboxy) and N(amino) terminal respectively, connected by a α-helix, with one iron binding site on each lobe. LF binds primarily Fe⁺²or Fe⁺³but is also capable of binding other metals ions traces like Al³⁺, Ga³⁺, Mn³⁺, Co³⁺, Cu²⁺, Zn²⁺but with much lower affinity [11].

LF is found primarily in exocrinesecretions such as milk [12,13] tears, nasal exudate or bronchial mucus [11,14-18], and is also known as a major component of the secondarygranules of neutrophils [19]. Conversely, blood, plasma and serum levels of LF are very low [16]. An increase of LF in blood may occur during infection, inflammation or tumor growth [20].

Due to its higher affinity for iron [21], the ability to retain iron over a broad pH range [22,23], and differential tissue relative distribution than other transferrins, LF own unique functional properties.

Following infection of inflammation, pH levels on sites of inflammation become very low (<4.5) due to metabolic activity of bacteria. In such a condition LF prevents bacterial proliferation by its bound to iron, also the one released from transferrin [24].

Being a multifunctional molecule, LF own several physiological functions: i) iron absorption and metabolism [25,26]; ii) as a part of the innate immune system, LF protects against microbial infections, both Gram-positive and negative bacteria, viruses, protozoa, or fungi [27]; iii) antibacterial [27] and antiviral activity [28]; stimulation of bone growth [29]; prevention of inflammation by reducing production of pro-inflammatory cytokines [30,31] and diminishing oxidative stress [32]; antiparasitic activity [33]; antitumoractivity [34-36].

Recently, LF has been named “Nutraceutical Protein”, due to its multiple properties and the potential for use as a therapeutic protein [37].

Until recently, the main source of LF was from human breast milk. Nowadays, however, LF from bovine source, Bovine Lactoferrin (bLF) is ready available.bLFhas about 69% amino acids identity with Human LF(hLF) [38] but, despite slight changes in domains orientation and closure any functional differences are found [39]. Therefore, the carbohydrate structure of bLF is much better defined than that of hLF [40] and it has been shown that bLF bind to humanneutrophils with higher affinity than hLF [41].

bLFis generally recognized as safe (GRAS) by the Food and Drug Administration (FDA) and permitted as food and dietary supplement ingredient in many countries. Supplements of bLF are reported to have the ability to support the immune system by anti-infective, anti-cancer, and anti-inflammatory effects [42] highlighting the bLF potential as therapeutic agent. 

Nowadays several studies have re-evaluated the role of dietary interventions in the developmentand therapy of skin disease. This led to an increasing interest in nutraceutical and dietary supplement as novel therapeutic agents. 

Due to its various and interesting functions, LF has attracted a growing interest for potential clinical applications and there are increasing evidences for its use in treatments of different dermatological conditions [43].

The aim of the present work was to study the effects of bLF on Lipopolysaccharide (LPS)-induced cytokinesexpression and secretion in two cell lines (human keratinocytes NCTC2544 and THP-1 myelomonocyticleukaemia cells) to deeper understand its potential in the treatment of skin related disease.

3. Materials and Methods 

3.1. Chemicals

Lactoferrin Moringa Low endotoxin bovine milk Lactoferrin (LF) (<1 EU/mg, <20% iron saturated, >95% purity) was provided by C.F.M. CO. Farmaceutica Milanese S.p.A. (Milan, Italy). Cell culture media and all supplements were from Lonza Inc. (Barcelona, Spain), for NCTC2544 cell and from Sigma (St Louis, MO, USA) for THP-1 cells. Bacterial Lipopolysaccharide (LPS) (Escherichia coli, Serotype 0111:B4, 3×10⁶EU/mg) and Elisa reagents were purchased from Sigma. Antibodies and protein for ELISA assay were from R&D System (Minneapolis, MN, USA).

3.2. Cell culture and Viability

Normal human keratinocyte NCTC 2544 (Istituto Nazionale di Ricercasul Cancro–Italy) were cultured under humidified atmosphere (5% CO2, 37^°C) on Eagle’s Minimum Essential Medium Balanced with salt solution (EMEM-EBSS) containing 2mM l-glutamine, 1% of Non-Essential Amino Acids (NEAA) and penicillin (100 U/ml)/streptomycin (100 U/ml) supplemented with 10% Fetal Bovine Serum (FBS) (basal medium). THP-1 cells (IstitutoZooprofilattico di Brescia, Bres-cia, Italy) were cultured in RPMI 1640 containing 2 mM l-glutamine, 0.1 mg/ml streptomycin, 100 IU/ml penicillin, 50µM-2-mercaptoethanol, supplemented with 10% heated-inactivated fetal calf serum (media). 

Both cell types were incubated in 25 cm² surface culture flasks at 37^°C with 5% CO₂ until ca. 80% of confluence was reached. Following harvesting with trypsin/EDTA cells were seeded at 5x10⁴ cells per well into 96 well plates or 1x10⁶ cells per well into 12 well plates for 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide proliferation (MTT) assay and cytokines quantification, respectively. 

3.3. Effect of bLF Preparations on Viability of Cells

Cytotoxicity of LF on NCTC2544 and THP-1 cell lines was analyzed by MTT assay according to the method of Hansen [43] with minor modifications. After reaching 80% confluence, cells were exposed to bLF at the following concentrations: 2.5, 5, 10, 20, 40, 80 and 160µg/mL, for 24h at 37^°C, under 5% of CO₂. Cells in basal medium alone were used as control. At the end of treatment, the medium was replaced with 100 μl per well of the MTT solution and cells were incubate for 3h in darkness, at 37^°C, under 5%. MTT was previously dissolved (5 mg/ml) in PBS and diluted 1:10 in the cell culture medium without phenol red. Following incubation, 100 μl per well of Dimethylsulphoxide (DMSO) were added to dissolve purple formazan product and the solution shaked for 15 min at room temperature. Finally, the absorbance of the solutions was read at 550 nm in a microplate reader (BioTek Instruments Inc., Bad Friedrichshall, Germany). Each experiment was carried out in triplicate. Data were expressed as the mean percentage of viable cells compared with control.

3.4. TNF-α, IL-10 and IL-12 Immunomodulation by bLF

Immunomodulatory properties of bLF were investigated first by mean of qRT-PCR. Total RNA was isolated at different times of treatment using a commercial available kit (TriReagent from Sigma) as described by Chomczynski and Mackey [45]. 2µg of total RNA were retro-transcribed in cDNA using a high-capacity cDNA kit from Applied Biosystems (Foster City, CA, USA) in a thermal cycler (Stratagene Mx3000P Real Time PCR System, Agilent Technologies Italia S.p.A., Milan, Italy) according to these conditions: 25^°C for 10 min, 37^°C for 120 min and 85^°C for 60 s. mRNA levels where then quantified by using Taq-Man TM-PCR technology. Following 20X TaqMan^® assay (Applied Biosystems) were used: Hs00174128-m1 (TumorNecrosis Factor Alpha, TNF-α), Hs00961622_m1 (Interleukin-10, IL-10), Hs01011518_m1 (Interleukin-12, IL-12) and Hs999999-m1 (Human glyceraldehyde-3-phosphate dehydrogenase, GAPDH). PCR amplifications were carried out using 40 ng of cDNA in a 20 μl of mixture reaction containing 10 μl of 2XPremix Ex Taq (Takara, Clontech Laboratories, Inc, Mountain View, USA), 1 μl of 20× TaqMan gene expression assay, 0.4 μl of 50X RoxTM reference dye II (Takara, Clontech Laboratories, Inc,), 4.6 μl of water and 4 μl of cDNA. PCR conditions were 95^°C for 30 secs (for Amplitaq activation) followed by 40 amplification cycles (95^°C for 5 s; 60^°C for 20sec). Analyses were carried out in triplicate. Average value of Human GAPDH gene was used as endogenous reference for target gene and the quantification of transcripts levels was performed by the 2^−ΔΔCTmethod [46].

The expression of cytokines at protein levels was also assessed in cell free supernatants, stored at -80^°C until measurement, after centrifugation at 1200 rpm for 5 mins. Before ELISA assay samples were concentrated with Vivaspin Sartorius centrifugal concentrators (with cut off 10000 and 30000 MW, respectively for IL-10 and IL-12/TNF-α). Protein expression was assessed by custom sandwich Elisa assays, according to manufacturer recommendations as regards antibodies and standards dilutions and following a custom home-made protocol for coating and detection of antibodies. Results were expressed as pg/mg protein.

3.5. Statistical Analysis

All experiments were repeated at least three times, with representative results shown. Data are expressed as Mean ±SEM for qRT-PCR analysis and Standard Deviation (SD) for other experiments. Results were checked for normal distribution using Shapiro-Wilk test before further analyses. Analysis of Variance (ANOVA) was carried out following by Sidak’s multiple comparison test, using Graph Pad Prism version 6.00 for Windows (Graph Pad Software, La Jolla California USA, www.graphpad.com). P-values equal to or less than 0.05 were considered significant.

4. Results 

4.1. In vitro Effects of bLF on TNF-α, IL-10 and IL-12 Anti-Inflammatory Cytokines

Primarily, lactoferrin 10 µg/mL (LF10) and 40 µg/mL (LF40) effect on LPS-induced cytokines gene expression was determined by mean of qRT-PCR. Down-regulation in cytokines expression reflects into an anti-inflammatory action. Both LFs exerted a significant (P<0.0001) immunomodulatory action on TNF-α since 1 to 8h of treatment (Figure 1a) respect to Positive Control (PC), when tested on human keratinocytes NCTC2544. On THP-1 cells (Figure 1b), even if treatment with both LFs produced a significant (P<0.0001) down-regulation of TNF-α after 1h of treatment, the strongest LF dose-independent immunomodulation was found from 5h to 7h of incubation. At 8h LF40 was most effective than LF10. In addition, IL-10 expression was strongly significant (P<0.0001) influenced by LFs (vs PC), independently from concentrations and in general, independently from cell line (Figure 2a and 2b). Most interesting, on THP-1 cells this effect was evident since 1h of incubation (Figure 2b).

On the contrary, immunomodulation of IL-12 cytokine was strongly dependent from cell line used. Both LFs showed to exert a similar significant (P<0.0001) anti-inflammatory action on NCTC2544 cells from 2h of incubation (Figure 3a). This effect wears off after 5h and 6h of incubation for LF40 and LF10, respectively. On THP–1 cells, immunomodulation by LFs respect to PC was less time and dose dependent (Figure 3b). The strongest significant (P<0.0001) effect was found for LF10 from 3 to 5h of incubation and LF40 after 7h. Statistical significance (p value) is reported in Table S1.

qRT-PCR results were mostly confirmed by ELISA assay and this was according to the delayed expression of a protein respect to the related gene. In particular, according to TNF-α gene expression, Elisa assay (Table 1) confirmed LFs anti-inflammatory activity, independently from cell line used. On NCTC2544 cells IL-10 protein expression was delayed at 6h of treatment and continued up to 8h. LF10 and LF40 produced the same anti-inflamamtory effect, respect to PC, with the exception of 7h treatment, following which LF40 was most effective than LF10 (Table 2). On THP-1, IL-10 expression was delayed above 8h of treatment (Table 2). Same results were found for IL-12 expression on NCTC2544 (Table 3).

On the contrary, according to gene expression evaluation, on THP-1 cell line, LFs immunomodulation was most evident from 6 to 7h of incubation (Table 3), independently from doses.

The inhibitory effects exerted by LFs on cytokine expression, following the LPS-mediated inflammation were not due to cytotoxicity as assessed by MTT assay (Table 4).

5. Discussion

bLFis an iron-binding glycoprotein that consists of a single polypeptide chain of 689 amino acids; the sequence homology with human LF is 69% [39].

As an integral part of the innate immune system LF is considered a well-known immunomodulator of leukocyte populations [47-52] and many recent evidences support the role of Lf in regulation of host acting by inhibition of several cytokines [30,53] and act primarily as key modulators and regulators of immune processes. Produced also by cells other than immune cells, after the binding to specific receptors, cytokines produce multiple signals and induce target cell to new mRNA and protein synthesis [54]. This results in a specific biological response.

Among cytokines, TNF-α [55-58], IL-10 [59-63], IL-12 [64–66], are key effectors both in immune and infectious response[67]. 

Studies on various human monocytic cell lines, included THP-1 cells, showed that LF, both bovine and human,can modulate basal and LPS-mediated pro-inflammatory cytokines release [31,68,69]. LPS is the major constituent of the outer membrane of bacterial pathogens and is a well-known initiator of inflammation [70]. Among others, Appelmelk and coworkers [71] suggested that, in systemic infections, the inhibition of primary inflammatory response TNF-α-linked could be due to the binding between LF and the lipid A moiety of LPS released from bacteria, inhibiting subsequent LPS-mediated pro-inflammatory response. 

LF, both human and bovine, present similar binding to THP-1 cells [72]. In a more recent study on THP-1 monocytes, Haversen [31] demonstrated that LF regulates the LPS-induced cytokine expression on a transcriptional level, by interference with the intracellular events leading to NF-kB activation. Therefore, in the same study, bLFseemed somewhat more efficient compared to human LF and that TNF-α, the most inhibited cytokine, down-regulates IL-10 expression. 

Moreover, many receptors have been identified both on the surfaces of immunocompetent cells [73-75] and epithelial cells [19] suggesting also a direct involvement of LF in the signaling pathways of pro-inflammatory cytokines. 

Inflammation playsan important role in pathogenesis of many cutaneous disorders such as for example psoriasis [76], atopic dermatitis [77], contact dermatitis [78], acne vulgaris [79,80], UV-induced inflammation [81-83].

Although there are still few studies evaluating LF usage for dermatological conditions, the reported studies encourage the use of LF for these purposes [43].

In a first explorative study, Muller and coworkers[84] demonstrated the efficacy and tolerability of oral bLFsupplementation in subjects with mild to moderate facial acne vulgaris. Previously, Kim and coworkers [85] showed the ameliorating effect of Lactoferrin-enriched fermented milk on acne vulgaris. LF exerted these effects probably due to its anti-bacterial and anti-inflammatory effects [86]. 

LF has also the potential to prevent UV-induced skin damage by the inhibition of UV-stimulated cytokines [87] and acting as a sacrificial scavenger for Reactive Oxygen Species (ROS) [88]. Most interesting, also topical exposure to LF is able to influence inflammatory responses acting on local production pro-inflammatory cytokines [89].

The in vitro experiment presented in this work confirmed the immunomodulatory activity of bLF, which exerted its action at a transcriptional level since to the early phase to 8h of treatment. The anti-inflammatory activity occurs by immune-modulation of cytokines expression in an LPS-mediated inflammatory systemon normal human keratinocytes and THP-1 cells. Most likely bLF exerted its effect both by inhibition of binding of lipopolysaccharide endotoxin to cells, as well as acting directly on cells cytokines production.

6. Conclusions

Our results add an important element to the knowledge on the anti-inflammatory potential of bovine LF in an LPS-mediated system. bLF is capable of immunomodulate pro-inflammatory cytokines at a transcriptional level. These findings encourage the use of bLF as immunomodulator agentin the treatment of different cutaneous disorder (eg. acne vulgaris) both as dietary supplement and topically administered.

However larger randomized clinical trials are necessary to better define its role and pharmacokinetic behaviorfor dermatological purposes.

7. Conflicts of Interest: R.F. serves as a consultant for Giuliani S.p.A. P.D. and M.B. are employed by Giuliani S.p.A.

8. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

9. Author Contributions: P.D. performed experiments and wrote the paper; M.B. performed experiments, analyzed the data, wrote the paper; S.E. review the paper; R.F. conceived and designed the experiments, review the paper.

Figure 1: Time course of Tumor Necrosis Factor-α (TNF-α) gene expression on NCTC2544 (a) and THP-1 cells (b), respectively. Cells were seeded at 10⁶ cells/well and, after reaching 80% confluency, treated for different times (1-8h) with LF10 (Lactoferrin 10µg/mL) ( ), LF40 (Lactoferrin 40µg/mL) ( ) or basal medium 2.5% FBS (positive control-PC) (  ). Simultaneously inflammation was induced by adding basal medium at 2.5% FBS with LPS (10µg/mL). Data are the means±SEM of three separate experiments, n = 3. Statistical analysis was performed with Sidak’s.

 

Figure 2:  Interleukin 10 (IL-10) gene expression on NCTC2544 (a) and THP-1 cells (b), respectively. Cells were seeded at 10⁶ cells/well and, after reaching 80% confluency, treated for different times (1-8h) with LF10 (Lactoferrin 10µg/mL) (  ), LF40 (Lactoferrin 40µg/mL) (  ) or basal medium 2.5% FBS (Positive Control-PC)

(  ). Simultaneously inflammation was induced by adding basal medium at 2.5% FBS with LPS (10µg/mL). Data are the means±SEM of three separate experiments, n = 3. Statistical analysis was performed with Sidak’s.

Figure 3: Interleukin 12 (IL-12) gene expression on NCTC2544 (a) and THP-1 cells (b), respectively.Cells were seeded at 106 cells/well and, after reaching 80% confluency, treated for different times (1-8h) with LF10 (Lactoferrin 10µg/mL) ( ), LF40 (Lactoferrin 40µg/mL) ( ) or basal medium 2.5% FBS (Positive Control-PC)

(  ). Simultaneously inflammation was induced by adding basal medium at 2.5% FBS with LPS (10µg/mL). Data are the means±SEM of three separate experiments, n = 3. Statistical analysis was performed with Sidak’s.

Cell line	Time	TNF-alpha
		PC	LF10	LF40
NCTC2544	1h	110.04±41.29a	0.00±7.75b	0.00±11.30b
	2h	588.75±14.85c	250.16±7.73d	274.71±16.95d
	3h	588.72±27.82e	83.84±13.08f	340.70±103.56g
	4h	614.64±56.46h	395.81±50.52i	345.98±5.21i
	5h	746.99±33.59j	570.56±28.10k	534.79±18.63k
	6h	1,303.43±41.26l	575.38±75.47m	776.19±69.08m
	7h	916.62±15.98n	709.62±148.83n	876.99±54.07n
	8h	964.28±27.54o	601.01±14.01p	601.44±81.34p
THP–1	1h	606.25±29.16q	347.69±24.54r	347.69±24.54r
	2h	542.64±2.29s	445.50±82.38s	531.47±118.42s
	3h	586.46±60.31t	633.83±47.18t	663.50±22.65t
	4h	654.80±42.97u	740.03±29.86u	741.39±41.98u
	5h	729.36±87.82v	680.03±12.26v	713.96±30.70v
	6h	896.85±7.22w	796.16±18.35x	922.55±43.55y
	7h	889.30±15.70z	963.07±24.35z	832.72±53.91z
	8h	1,296.04±29.07aa	916.66±14.88ab	777.08±71.85ab

Table 1: Tumor Necrosis Factor-α (TNF-α) protein expression as determined by ELISA. Cells were seeded at 10⁶ cells/well and, after reaching 80% confluency, treated with LF10 (Lactoferrin 10µg/mL), LF40 (Lactoferrin 40µg/mL) or basal medium 2.5% FBS (Positive Control-PC). Simultaneously inflammation was induced by adding basal medium at 2.5% FBS with LPS (10µg/mL). Analyses were carried on collected cell free supernatant after incubation at 37^°C for 1-8h, under 5% CO₂. Data are the means±SD of three separate experiments, n = 3. Statistical analysis was performed with Sidak’s multiple comparison test. N.d. = Not Detectable. a-abValues with different superscript letters, differ significantly (P < 0.001).

 

Cell line	Time	IL–10
		PC	LF10	LF40
NCTC2544	1h	n.d.	n.d.	n.d.
	2h	n.d.	n.d.	n.d.
	3h	n.d.	n.d.	n.d.
	4h	n.d.	n.d.	n.d.
	5h	n.d.	n.d.	n.d.
	6h	53.17±1.87a	21.94±0.83b	12.92±1.31b
	7h	49.42±3.81c	8.15±9.99d	2.79±2.07e
	8h	60.17±2.96f	13.49±8.74g	5.24±0.60g
THP–1	1h	n.d.	n.d.	n.d.
	2h	n.d.	n.d.	n.d.
	3h	n.d.	n.d.	n.d.
	4h	n.d.	n.d.	n.d.
	5h	n.d.	n.d.	n.d.
	6h	n.d.	n.d.	n.d.
	7h	n.d.	n.d.	n.d.
	8h	n.d.	n.d.	n.d.

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