Honey is used as a therapy to aid wound healing. Previous data indicate that honey can stimulate cytokine production from human monocytes. The present study further examines this phenomenon in manuka honey. As inflammatory cytokine production in innate immune cells is classically mediated by pattern recognition receptors in response to microorganisms, bacterial contamination of honey and the effect of blocking TLR2 and -4 on stimulatory activity were assessed. No vegetative bacteria were isolated from honey; however, bacterial spores were cultured from one-third of samples, and low levels of LPS were detected. Blocking TLR4 but not TLR2 inhibited honey-stimulated cytokine production significantly. Cytokine production did not correlate with LPS levels in honey and was not inhibited by polymyxin B. Further, the activity was reduced significantly following heat treatment, indicating that component( s) other than LPS are responsible for the stimulatory activity of manuka honey. To identify the component responsible for inducing cytokine production, honey was separated by molecular weight using microcon centrifugal filtration and fractions assessed for stimulatory activity. The active fraction was analyzed by MALDI-TOF mass spectroscopy, which demonstrated the presence of a number of components of varying molecular weights. Additional fractionation using miniaturized, reverse-phase solidphase extraction resulted in the isolation of a 5.8- kDa component, which stimulated production of TNF-
via TLR4. These findings reveal mechanisms and components involved in honey stimulation of cytokine induction and could potentially lead to the development of novel therapeutics to improve wound healing for patients with acute and chronic wounds.

Introduction

Honey has been used traditionally in wound dressings for thousands of years. The treatment has regained popularity in recent times as an adjunct therapy to improve wound healing. A number of small clinical trials have been completed, which indicated that topical application of honey to wounds clinically improved wound healing and reduced healing times and scarring. Understanding the scientific basis of these effects could potentially lead to the development of novel therapeutic agents for the treatment of acute and chronic wounds. To date, much of the research associated with honey and wound healing has concentrated on the effects of Manuka honey, which is produced from nectar collected from Leptospermum scoparium, which grows wild in New Zealand. It is a complex mixture of carbohydrates, fatty acids, proteins and amino acids, vitamins, and minerals. Active Manuka honey is renowned for its antibacterial activity, and batches are assigned a unique Manuka factor (UMF) value corresponding to antibacterial activity (e.g., UMF 20 has equivalent antibacterial activity to 20% phenol w/v).

Normal wound healing is a complex process in which damaged tissue is removed and gradually replaced by restorative tissue during an overlapping series of events, which include coagulation, inflammation, cell proliferation, and tissue remodeling. The inflammatory phase of healing has an essential role in clearing the wound site of infectious agents and debris; this is facilitated by the activities of innate immune cells such as neutrophils and macrophages, which migrate to the wound site in response to tissue damage. These cells aid the resolution of infection and removal of foreign material and cellular debris by phagocytosis. The individual role of neutrophils and macrophages has been investigated, and previous studies indicate that macrophages have an essential role in wound resolution, as the absence of macrophages leads to poor debridement of the wound site and delayed repair. In contrast, depletion of neutrophils leads to enhanced wound closure. In addition to their phagocytic role, macrophages release various growth factors and cytokines, which are important in perpetuating the healing process. Recent studies indicate that production of IL-6 and TNF by macrophages and other cells at the wound site is essential in the healing process. We have shown previously that a variety of honey types can stimulate human monocytic cells to produce inflammatory cytokines (e.g., TNF, IL-6) important in resolution of infection and tissue repair. However, the components of honey responsible for this modulatory effect and the mechanism of action are yet to be determined. Previous studies have indicated that honey samples may be adulterated with microorganisms, including spore-forming aerobic and anaerobic bacteria. The presence of microorganisms or their cellular components could possibly explain the immune-stimulatory activity of honey. Innate immune cells such as monocytes and macrophages produce inflammatory mediators in response to the presence of microbes following engagement of microbial components with pattern recognition receptors (PRR) expressed by the cells; these include TLRs. This study demonstrates that the observed effects of honey on cytokine production in myeloid cells are not a consequence of bacterial contamination of honey or LPS (a.k.a., endotoxin) but are specifically associated with a 5.8-kDa moiety isolated from Manuka honey. Furthermore, this component stimulates inflammatory responses in monocytes via interactions with TLR4.

Materials and Methods

Cell Culture

The effect of honey or honey components on inflammatory cytokine production was assessed in primary human monocytes, MonoMac6 cells (MM6), or murine bone marrow-derived macrophages (BMDMs) from wild-type C57BL/6 mice or TLR2 or TLR4 knock out (KO) mice. Monocytes were obtained from peripheral blood from healthy volunteers. Polymorphonuclear cells were isolated by density gradient centrifugation, and monocytes were enriched using the mini-MACS monocyte negative selection kit according to the manufacturer’s instructions. Monocytes were cultured in RPMI-1640 medium, supplemented with 10% heat-inactivated FBS, 1% 2 mM L-glutamine, 1% nonessential amino acids, 1% penicillin (50 IU/ml)/streptomycin (100g/ml), and 1% sodium pyruvate at 37°C in a 5% CO2-humidified atmosphere. The human monocytic cell line MM6 was obtained from the German collection of microorganisms and cell cultures. MM6 cells were maintained in the same medium as primary monocytes. Cells were subcultured every 3 days at a density of 0.4 106 cells/ml. To assess the role of TLRs in cellular responses to manuka honey, BMDMs obtained from wild-type C57BL/6 and TLR2 and TLR4 KO mice were examined. BMDMs were incubated in the presence or absence of 1% w/v honey. BMDMs were isolated from 6- to 8-week-old mice and cultured as described previously.

Preparation of Honey-Supplemented Media

Fifteen different batches of New Zealand Manuka honey were used throughout the study. The honey samples were from known floral sources and assessed for their antimicrobial activity by a Staphylococcus aureus (ATCC 25923) inhibition assay, and UMF values were assigned accordingly. A control sugar syrup (artificial honey) was prepared as described previously. Honey solutions were made up to 1% (w/v) in supplemented medium and rendered sterile by filtration (0.45 M).

Bacterial Content of Honey Samples

All honey samples were assessed for the presence of viable bacteria and spores under aerobic and anaerobic conditions. Honey samples were spread directly onto blood agar or following enrichment for 5 days in cooked meat broth or Hartley’s digest broth before culture on blood agar.

LPS (Endotoxin) Content of Honeys

All honey samples were assessed for LPS content using the kinetic Limulus amoebocyte lysate assay (KQCL).

Cellular Viability of Cells Incubated with Honey

MM6 cells at a density of 1 x 10 cells/ml were incubated with 1% (w/v) of each honey solution for 0–24 h. The cells were washed in PBS (x3) and resuspended in 1 ml fresh media. Assessment of cellular viability was determined using trypan blue or the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)- 2-(4-sulfophenyl)-2H-tetrazolium bioreduction assay as described previously. Cell viability remained above 90% for all samples tested at all time-points assessed.

Measurement of TNF-x, IL-1B, or IL-6 Release from Human Monocytes or Murine Myeloid Cells

To determine the effect of honey or honey fractions on cytokine release, 1 x 10 cells/ml were incubated with 1% (w/v) honey, individual honey fractions, or heat-treated honey for 4 h (TNF-
production) or 24 h (IL1-B and IL-6 production) at 37°C in 5% CO2 atmosphere. Following incubation, supernatants were collected and stored at – 80°C. TNF-x, IL-1B, or IL-6, in cell culture supernatants, were quantified by ELISA in accordance with the manufacturer’s instructions.

Determining the Effect of Polymyxin B on Honey-Stimulated Cytokine Release

To assess the role of LPS in honey-mediated cytokine release, MM6 cells were incubated with polymyxin B, a chelator of LPS, prior to honey treatment. MM6 cells were incubated with polymyxin B (10 ml) for 1 h before addition of 1% honey samples or LPS (100 ng/ml) for 4 or 12 h. Following incubation, supernatants were collected and stored at –80°C. TNF-x, IL-1B, or IL-6 in cell culture supernatants were quantified by ELISA in accordance with the manufacturer’s instructions.

Separation of Honey Components by Microcon Centrifugal Filtration, Gel Filtration, and Dialysis

To begin to identify the active components of Manuka honey, samples were fractionated according to molecular weight. Initially, Manuka honey was fractionated by three different methods to determine the approximate mass of the component(s) responsible for cytokine stimulation. Honey samples were first subjected to dialysis using membranes with molecular weight cut-off (MWCO) of 3, 8, and 14 kDa. Alternatively, honey samples were fractionated sequentially using microcon centrifugal filters to provide fractions with apparent molecular weights of <3, 3–10, 10–30, and >30 kDa. Honey was also fractionated by gel filtration using Sephadex G-25 column. Component elution was carried out with standard RPMI medium at a flow rate of 0. 2 ml/min. Fractions equivalent to 1% total honey (w/v) were assessed for their ability to stimulate TNF-x, IL-1B, or IL-6 synthesis in MM6 cells (see above). Fractions were freeze-dried for MALDI-TOF analysis and further.

MALDI-TOF Mass Spectrometry (MS)

Freeze-dried honey fractions were reconstituted in water and mixed in a 1:10 sample:matrix ratio with Sinapinic acid matrix solution [10 mg/ml sinapinic acid in 70/30 0.1% aqueous trifluoroacetic acid (TFA)/acetonitrile (ACN)]. The resulting mixture was applied to a MALDI plate and allowed to air dry. The plate was then inserted into a Voyager DE-STR MALDI-TOF MS and spectra acquired between 1000 and 15,0000 amu in positive ionization linear mode using an acceleration voltage of 25 kV, a grid voltage of 90% of the acceleration voltage, a delay time of 750 ns, and 100 laser shots per spectrum.

Miniaturized RP-SPE Fractionation

The sample was dissolved in 10 l 0.1% TFA. A C18 RP ziptip (Millipore, UK) was conditioned with five washes of 50/50 methanol/0.1% TFA followed by five washes with 0.1% TFA before sample application. The sample was applied to the ziptip and allowed to wash over the packing 10 times; therefore, the sample solution contained only the components not bound to the ziptip, which was washed five times with 0.1% TFA before elution using 80/20 ACN/0.1% TFA. Both fractions were freeze-dried prior to bioassay.

Monosaccharide Composition Analysis

The isolated, 5.8-kDa component was assessed for the presence of monosaccharide. Briefly, the internal standard (Arabitol) was added to each sample and lyophilized. The sample was then subjected to methanolysis in 1 N methanolic/ HCl (80°C for 16 h under nitrogen) followed by derivatization and analysis by gas chromatography-MS.

Amino Acid Composition Analysis

The isolated, 5.8-kDa component was assessed for the presence of amino acids. Briefly, the internal standard (Norleucine) was added to each sample and hydrolyzed in 6 N HCl for 4 h at 145°C. The samples were derivatized and analyzed by RP-HPLC coupled with UV detection. The data were then compared with that obtained from analysis of a standard mixture containing 50 nmoles each amino acid and 50 nmoles internal standard.

Blocking of PRR TLR2- and -4-Mediated Responses

To assess the role of PRR in cellular responses to manuka honey, cytokine production was assessed in the presence of anti-TLR2 and anti-TLR4 antibodies. TLR2 and TLR4 receptors were blocked on the surface of MM6 cells or primary human monocytes prior to incubation with 1% (w/v) honey or fraction solutions. Monocytes were incubated with 10 g/ml anti-TLR2 or 20 g/ml anti-TLR4, respectively, for 1 h prior to addition of honey or fraction samples to give a final concentration of 1% (w/v) honey. Cells were then incubated overnight and assayed for TNF-
or IL-6 as described above. To control for nonspecific binding, an IgG2a isotype control antibody was used. Experimental conditions were optimized previously for maximum inhibition of specific ligand- stimulated cytokine responses for TLR2 and TLR4, lipoteichoic acid, and LPS, respectively.

Results

Characterization of Manuka Honey

Human monocytic cells, MM6, were incubated with different batches of 1% (w/v) Manuka honey to profile their effect on cytokine production. In accordance with our previous studies, Manuka honey stimulated the production of inflammatory cytokines TNF-x, IL-1B, or IL-6 in this cell line and in human peripheral blood monocytes (data not shown). Each batch of Manuka honey was assessed for bacterial contamination and the presence of spores under aerobic and anaerobic conditions. All samples were negative for vegetative bacterial growth. However, from just over one-third of honey samples (6/15), aerobic and/or anaerobic spores were recovered, and the majority was identified as Bacillus species. Further, when LPS content was assessed, only low levels of LPS (0.27 ng/ml) were detected in each batch of honey. To assess the stimulatory activity of equivalent LPS concentrations, MM6 cells were stimulated with 1 ng/ml LPS, and cytokine production was assessed. Stimulation with this concentration of LPS resulted in low levels of cytokine production, ten-, four-, and threefold less synthesis of IL-1B, IL-6, and TNF-x, respectively, when compared with cells treated with 1% (w/v) honey. As a further control, sugar syrup was shown not to stimulate significant cytokine production under identical culture conditions. Although stimulation of inflammatory responses by the honey samples varied slightly from batch to batch, there was no correlation of stimulation with bacterial spore or LPS concentration. Further, Manuka samples were assessed for antibacterial activity and assigned a UMF value accordingly. The antibacterial activity of the samples ranged from equivalent to 5–24.4% (v/v) phenol but was not associated with cytokine production.

Honey-stimulated cytokine production is mediated via interactions with TLR4 but not TLR2

Innate immune cells respond to the presence of microbes, debris, and foreign material via PRR, which include the TLRs. Of the TLRs identified to date, TLR2 and TLR4 are amongst the best-characterized with regard to their specificity and downstream signaling pathways. TLR2 recognizes lipoproteins/lipopeptides and peptidoglycan, whereas TLR4 recognizes LPS from Gram-negative bacteria [26]; however, numerous other exogenous and endogenous ligands have been identified for these receptors [27]. To investigate whether Manuka honey stimulates myeloid cells via TLR2 or -4, MM6 cells were preincubated with antibodies directed against the ligand-binding domain of TLR2 or TLR4. Treatment of cells with anti-TLR2 antibody did not affect cytokine production by cells stimulated with honey (data not shown). However, anti-TLR4-blocking antibody significantly inhibited honey-induced TNF-x production by 70%. These data suggest that the active component(s) of honey signal through TLR4 but not TLR2. This was confirmed further in BMDMs isolated from TLR2 and TLR4 KO mice. Following stimulation with 1% (w/v) honey, wild-type and TLR2 KO murine macrophages produced TNF-x in a similar manner to that observed in human monocytic cells. However, BMDMs derived from TLR4 KO mice did not produce significant levels of TNF-x in response to honey incubation.

Fractionation of manuka honey indicates that it contains an active component of 5.8 kDa

To determine the component(s) of honey responsible for inducing cytokine production in myeloid cells, gel fractionation and dialysis were performed initially. Preliminary data indicated that a component of 5–6 kDa present in honey was able to stimulate TNF-x production. These methodologies proved problematic for downstream applications, namely associated with dilution/contamination and loss of active components, respectively. Further studies using microcon centrifugal filtration allowed concentration of the active fractions and control of dilution. Using this method, the majority of cytokine-stimulatory activity was found to be associated with fractions with an apparent molecular weight greater than 30 kDa, although some activity was present in the 3-kDa fraction. To further characterize the chemistry of these components, honey and its fractions were heat-treated prior to incubation with MM6 cells. Heat treatment of unfractionated honey caused a significant reduction in the ability of honey to stimulate IL-1B, IL-6, or TNF-x production in MM6 cells. A similar effect was observed in the fractionated honey. Taken together, the data indicate that the active components are heat-sensitive and provide further evidence that components other than LPS (a heat-stable molecule) are responsible for the activity associated with Manuka honey. Therefore, further analysis was performed by MALDI-TOF MS.

MALDI-TOF analysis was restricted to the 30-kDa fraction, as the majority of cytokine-stimulatory activity was associated with these components. This strategy demonstrated the presence of a small number of high molecular weight components. It is more surprising that a number of less than 30 kDa molecular weight moieties were also observed in this fraction, possibly as a consequence of binding of these components to larger molecules. There was a variation in the peaks present across the samples and their relative proportions. As gel filtration and dialysis indicated that the active component had a mass of 5–6 kDa, this peak was purified further using miniaturized RP-SPE separation, and purity was confirmed by MALDI-TOF analysis, and its ability to stimulate cytokine production was assessed. This component was found to stimulate TNF-x production in monocytes, whereas a fraction containing the remaining components determined to be present in the 30-kDa fraction did not stimulate the production of this cytokine (data not shown). In experiments assessing the activity of the isolated component, a number of samples isolated from different batches of honey were used to ensure that the data were representative of the different batches. There was some variation in activity associated with different batches; however, all batches stimulated similar levels of cytokine induction.

The isolated component was examined for the presence of amino acids and monosaccharides. Analysis revealed the absence of amino acids from the isolated component, indicating that the component is not a protein. Monosaccharide analysis revealed the presence of monosaccharides, but further analysis is required to determine whether this represents contamination with low molecule weight sugars or indicates that the component contains complex oligosaccharide.

As unfractionated honey stimulated TNF-x production via TLR4, we assessed the role of the 5.8-kDa component in stimulating production of this cytokine via this receptor. As shown in Figure 5, TNF-x production stimulated by the 5.8- kDa component was abrogated in MM6 cells by pretreatment with anti-TLR4. These data were confirmed in primary human monocytes, as illustrated in Figure 6. TNF-x production stimulated by the 30-kDa fraction or the purified 5.8-kDa component was inhibited significantly in primary human monocytes following pretreatment with anti-TLR4 antibody. In addition, when BMDMs from wild-type and TLR4 KO mice were incubated in the presence of the isolated component, TNF-
production was depressed significantly in TLR4 KO compared with wild-type or TLR2 KO BMDMs (Fig. 7). Taken together, these data suggest that Manuka honey contains a heat-sensitive, 5.8-kDa component, which stimulates cytokine production via TLR4, and that this activity is not associated with bacterial endotoxin.